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Nishiyama S, Takemoto Y, Yamanouchi K, Kondo K, Kawatsu S, Maruyama M, Higaki K. Dynamic changes in the distribution equilibrium of drugs in microemulsions associated with drug absorption facilitate the absorption improvement for drugs with low water-solubility by self-microemulsifying drug delivery system (SMEDDS). Int J Pharm 2025; 674:125458. [PMID: 40074161 DOI: 10.1016/j.ijpharm.2025.125458] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 02/28/2025] [Accepted: 03/08/2025] [Indexed: 03/14/2025]
Abstract
Mechanisms for absorption improvement of drugs with low water-solubility by self-microemulsifying drug delivery system (SMEDDS) are still controversial except for solubility improvement. We attempted to clarify the mechanisms by utilizing model drugs classified as biopharmaceutics classification system class II. In the in-vitro transport study for microemulsions (MEs) formed from SMEDDS, the permeation clearance (CLperm,freeSMEDDS) calculated based on free drug concentrations in MEs, was significantly larger than the CLpermsoln for aqueous solution. However, pretreatment of intestinal mucosa with drug-free MEs did not change CLpermsoln so much. The contribution of endocytosis to drug absorption from MEs was negligible. Instead, our novel egg phosphatidylcholine-monolayer-chloroform partition study revealed that drugs were continuously released from ME droplets, and that the distribution equilibrium of drugs in ME dynamically shifted from ME droplets to aqueous phase associated with their partitioning into chloroform phase (i.e. drug absorption). CLperm,freeSMEDDS did not reflect the continuous drug release or the much larger amount of drugs available for absorption than revealed as free concentrations and thereby overestimated the permeation clearance. The absorption improvement by SMEDDS could be attributed to the dynamic changes in the distribution equilibrium of drugs in MEs associated with drug absorption, i.e., the continuous drug release from ME droplets.
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Affiliation(s)
- Saki Nishiyama
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Yuki Takemoto
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan; Preformulation Group1, Formulation Development Department, Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 216 Totsuka-cho, Totsuka-ku, Yokohama, Kanagawa 244-8602, Japan
| | - Keita Yamanouchi
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Keiji Kondo
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan; Preformulation Research Laboratory, CMC Headquarters, Otsuka Pharmaceutical Co., Ltd. 224-18 Hiraishi Ebisuno, Kawauchi-cho, Tokushima 771-0182, Japan
| | - Sho Kawatsu
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan; Formulation Design, Pharmaceutical Research and Technology Laboratories, Pharmaceutical Technology, Astellas Pharma Inc., 180 Ozumi, Yaizu, Shizuoka 425-0072, Japan
| | - Masato Maruyama
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Kazutaka Higaki
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
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Li R, Tang Y, Huang W, Li R, Liu J. The Roles of Apolipoprotein A1-Binding Protein in Metabolic Diseases. Nutr Rev 2025:nuaf021. [PMID: 40036350 DOI: 10.1093/nutrit/nuaf021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/06/2025] Open
Abstract
Metabolic disorders, including atherosclerosis, diabetes, and metabolic dysfunction-associated steatotic liver disease, are closely related to increased cardiovascular risks, significantly harming human life and health. Apolipoprotein A1-binding protein (AIBP), a multifunctional protein, plays crucial role in cholesterol metabolism. AIBP exerts an important action in managing metabolic diseases by interacting with apolipoprotein A-I and ATP-binding cassette transporter A1 activities to regulate high-density lipoprotein)-mediated cholesterol transport and to maintain lipid homeostasis. In addition, AIBP suppresses inflammatory stress and abnormal angiogenesis, and acts as an NAD(P)HX epimerase to optimize energy metabolism. In this review, the multiple roles of AIBP in clinical metabolic diseases are summarized, and AIBP is proposed to be a potential therapeutic target against metabolic diseases.
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Affiliation(s)
- Ruihan Li
- Key Laboratory of Environmental Pollution and Integrative Omics, Guilin Medical University, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin 541199, PR China
| | - Yuqi Tang
- Key Laboratory of Environmental Pollution and Integrative Omics, Guilin Medical University, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin 541199, PR China
| | - Wenjun Huang
- Key Laboratory of Environmental Pollution and Integrative Omics, Guilin Medical University, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin 541199, PR China
| | - Rong Li
- Key Laboratory of Environmental Pollution and Integrative Omics, Guilin Medical University, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin 541199, PR China
| | - Jiaqi Liu
- Key Laboratory of Environmental Pollution and Integrative Omics, Guilin Medical University, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin 541199, PR China
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Kraus A, Kratzer B, Sehgal ANA, Trapin D, Khan M, Boucheron N, Pickl WF. Macropinocytosis Is the Principal Uptake Mechanism of Antigen-Presenting Cells for Allergen-Specific Virus-like Nanoparticles. Vaccines (Basel) 2024; 12:797. [PMID: 39066435 PMCID: PMC11281386 DOI: 10.3390/vaccines12070797] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2024] [Revised: 06/29/2024] [Accepted: 07/12/2024] [Indexed: 07/28/2024] Open
Abstract
Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.
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Affiliation(s)
- Armin Kraus
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Bernhard Kratzer
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Al Nasar Ahmed Sehgal
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Doris Trapin
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Matarr Khan
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Nicole Boucheron
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
| | - Winfried F. Pickl
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria
- Karl Landsteiner University of Health Sciences, 3500 Krems, Austria
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Bannunah A, Cavanagh R, Shubber S, Vllasaliu D, Stolnik S. Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles. Mol Pharm 2024; 21:3603-3612. [PMID: 38864426 PMCID: PMC11220748 DOI: 10.1021/acs.molpharmaceut.4c00333] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Revised: 06/07/2024] [Accepted: 06/07/2024] [Indexed: 06/13/2024]
Abstract
Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.
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Affiliation(s)
- Azzah Bannunah
- School
of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
| | - Robert Cavanagh
- School
of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
| | - Saif Shubber
- School
of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
| | - Driton Vllasaliu
- School
of Cancer & Pharmaceutical Sciences, Faculty of Life Sciences
& Medicine, King’s College London,
Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K.
| | - Snow Stolnik
- School
of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
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Yung C, Zhang Y, Kuhn M, Armstrong RJ, Olyaei A, Aloia M, Scottoline B, Andres SF. Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid. J Extracell Vesicles 2024; 13:e12422. [PMID: 38602306 PMCID: PMC11007820 DOI: 10.1002/jev2.12422] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Accepted: 02/20/2024] [Indexed: 04/12/2024] Open
Abstract
Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.
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Affiliation(s)
- Claire Yung
- Department of PediatricsPediatric GI Division, School of Medicine, Oregon Health and Science UniversityPortlandOregonUSA
| | - Yang Zhang
- Department of PediatricsPediatric GI Division, School of Medicine, Oregon Health and Science UniversityPortlandOregonUSA
| | - Madeline Kuhn
- Department of PediatricsPediatric GI Division, School of Medicine, Oregon Health and Science UniversityPortlandOregonUSA
| | - Randall J. Armstrong
- Knight Cancer InstituteOregon Health and Science UniversityPortlandOregonUSA
- Cancer Early Detection Advanced Research (CEDAR)Oregon Health and Science UniversityPortlandOregonUSA
| | - Amy Olyaei
- Division of Neonatology, Department of PediatricsOregon Health and Science UniversityPortlandOregonUSA
| | - Molly Aloia
- Division of Neonatology, Department of PediatricsOregon Health and Science UniversityPortlandOregonUSA
| | - Brian Scottoline
- Department of PediatricsPediatric GI Division, School of Medicine, Oregon Health and Science UniversityPortlandOregonUSA
- Division of Neonatology, Department of PediatricsOregon Health and Science UniversityPortlandOregonUSA
| | - Sarah F. Andres
- Department of PediatricsPediatric GI Division, School of Medicine, Oregon Health and Science UniversityPortlandOregonUSA
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6
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Yung C, Zhang Y, Kuhn M, Armstrong RJ, Olyaei A, Aloia M, Scottoline B, Andres SF. Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.09.03.556067. [PMID: 38187651 PMCID: PMC10769189 DOI: 10.1101/2023.09.03.556067] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2024]
Abstract
Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.
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7
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da Costa Marques R, Hüppe N, Speth KR, Oberländer J, Lieberwirth I, Landfester K, Mailänder V. Proteomics reveals time-dependent protein corona changes in the intracellular pathway. Acta Biomater 2023; 172:355-368. [PMID: 37839632 DOI: 10.1016/j.actbio.2023.10.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Revised: 10/04/2023] [Accepted: 10/06/2023] [Indexed: 10/17/2023]
Abstract
The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. STATEMENT OF SIGNIFICANCE: Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers.
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Affiliation(s)
- Richard da Costa Marques
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany; Dermatology Clinic, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
| | - Natkritta Hüppe
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
| | - Kai R Speth
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany; Dermatology Clinic, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
| | - Jennifer Oberländer
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany; Dermatology Clinic, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
| | - Ingo Lieberwirth
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
| | - Katharina Landfester
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
| | - Volker Mailänder
- Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany; Dermatology Clinic, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany.
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8
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He M, Li Z, Tung VSK, Pan M, Han X, Evgrafov O, Jiang XC. Inhibiting Phosphatidylcholine Remodeling in Adipose Tissue Increases Insulin Sensitivity. Diabetes 2023; 72:1547-1559. [PMID: 37625119 PMCID: PMC10588299 DOI: 10.2337/db23-0317] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 08/16/2023] [Indexed: 08/27/2023]
Abstract
Cell membrane phosphatidylcholine (PC) composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT); changes in membrane PC saturation are implicated in metabolic disorders. Here, we identified LPCAT3 as the major isoform of LPCAT in adipose tissue and created adipocyte-specific Lpcat3-knockout mice to study adipose tissue lipid metabolism. Transcriptome sequencing and plasma adipokine profiling were used to investigate how LPCAT3 regulates adipose tissue insulin signaling. LPCAT3 deficiency reduced polyunsaturated PCs in adipocyte plasma membranes, increasing insulin sensitivity. LPCAT3 deficiency influenced membrane lipid rafts, which activated insulin receptors and AKT in adipose tissue, and attenuated diet-induced insulin resistance. Conversely, higher LPCAT3 activity in adipose tissue from ob/ob, db/db, and high-fat diet-fed mice reduced insulin signaling. Adding polyunsaturated PCs to mature human or mouse adipocytes in vitro worsened insulin signaling. We suggest that targeting LPCAT3 in adipose tissue to manipulate membrane phospholipid saturation is a new strategy to treat insulin resistance. ARTICLE HIGHLIGHTS
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Affiliation(s)
- Mulin He
- Department of Cell Biology, The State University of New York Downstate Health Sciences University, Brooklyn, NY
| | - Zhiqiang Li
- Department of Cell Biology, The State University of New York Downstate Health Sciences University, Brooklyn, NY
| | - Victoria Sook Keng Tung
- Department of Cell Biology, The State University of New York Downstate Health Sciences University, Brooklyn, NY
| | - Meixia Pan
- Lipidomics Core, The University of Texas Health Science Center at San Antonio, San Antonio, TX
| | - Xianlin Han
- Lipidomics Core, The University of Texas Health Science Center at San Antonio, San Antonio, TX
| | - Oleg Evgrafov
- Department of Cell Biology, The State University of New York Downstate Health Sciences University, Brooklyn, NY
| | - Xian-Cheng Jiang
- Department of Cell Biology, The State University of New York Downstate Health Sciences University, Brooklyn, NY
- Molecular and Cellular Cardiology Program, Veterans Affairs New York Harbor Healthcare System, New York, NY
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Knaack DA, Chang J, Thomas MJ, Sorci-Thomas MG, Chen Y, Sahoo D. Scavenger receptor class B type I is required for efficient glucose uptake and metabolic homeostasis in adipocytes. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.21.554190. [PMID: 37662321 PMCID: PMC10473602 DOI: 10.1101/2023.08.21.554190] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Obesity is a worldwide epidemic and places individuals at a higher risk for developing comorbidities that include cardiovascular disease and type 2 diabetes. Adipose tissue contains adipocytes that are responsible for lipid metabolism and reducing misdirected lipid storage. Adipocytes facilitate this process through insulin-mediated uptake of glucose and its subsequent metabolism into triglycerides for storage. During obesity, adipocytes become insulin resistant and have a reduced ability to mediate glucose import, thus resulting in whole-body metabolic dysfunction. Scavenger receptor class B type I (SR-BI) has been implicated in glucose uptake in skeletal muscle and adipocytes via its native ligands, apolipoprotein A-1 and high-density lipoproteins. Further, SR-BI translocation to the cell surface in adipocytes is sensitive to insulin stimulation. Using adipocytes differentiated from ear mesenchymal stem cells isolated from wild-type and SR-BI knockout (SR-BI -/- ) mice as our model system, we tested the hypothesis that SR-BI is required for insulin-mediated glucose uptake and regulation of energy balance in adipocytes. We demonstrated that loss of SR-BI in adipocytes resulted in inefficient glucose uptake regardless of cell surface expression levels of glucose transporter 4 compared to WT adipocytes. We also observed reduced glycolytic capacity, increased lipid biosynthesis, and dysregulated expression of lipid metabolism genes in SR-BI -/- adipocytes compared to WT adipocytes. These results partially support our hypothesis and suggest a novel role for SR-BI in glucose uptake and metabolic homeostasis in adipocytes.
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Honsho M, Fujiki Y. Regulation of plasmalogen biosynthesis in mammalian cells and tissues. Brain Res Bull 2023; 194:118-123. [PMID: 36720320 DOI: 10.1016/j.brainresbull.2023.01.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2022] [Revised: 01/08/2023] [Accepted: 01/27/2023] [Indexed: 01/29/2023]
Abstract
Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. Synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of patients with deficiency in peroxisome biogenesis suggests that de novo synthesis of plasmalogens contributes significantly to plasmalogen homeostasis in humans. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. Dysregulation of plasmalogen synthesis impairs cholesterol synthesis in cells and brain, resulting in the reduced expression of genes such as mRNA encoding myelin basic protein, a phenotype found in the cerebellum of plasmalogen-deficient mice. In this review, we summarize the current knowledge of molecular mechanisms underlying the regulation of plasmalogen biosynthesis and the link between plasmalogen homeostasis and cholesterol biosynthesis, and address the pathogenesis of impaired plasmalogen homeostasis in rodent and humans.
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Affiliation(s)
- Masanori Honsho
- Department of Neuroinflammation and Brain Fatigue Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
| | - Yukio Fujiki
- Institute of Rheological Functions of Food-Kyushu University Collaboration Program, Kyushu University, Fukuoka, Japan; Graduate School of Science, University of Hyogo, Hyogo, Japan.
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11
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Liu S, Zhang Q, He H, Yi M, Tan W, Guo J, Xu B. Intranuclear Nanoribbons for Selective Killing of Osteosarcoma Cells. Angew Chem Int Ed Engl 2022; 61:e202210568. [PMID: 36102872 PMCID: PMC9869109 DOI: 10.1002/anie.202210568] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Indexed: 01/26/2023]
Abstract
Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L- or D-phosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance.
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Affiliation(s)
- Shuang Liu
- School of Materials Science and Engineering, Wuhan University of Technology, 122 Luoshi Road, Wuhan, Hubei, 430070, China
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Qiuxin Zhang
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Hongjian He
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Meihui Yi
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Weiyi Tan
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Jiaqi Guo
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
| | - Bing Xu
- Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA, 02454, USA
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Yu W, Wang Z, Yu X, Zhao Y, Xie Z, Zhang K, Chi Z, Chen S, Xu T, Jiang D, Guo X, Li M, Zhang J, Fang H, Yang D, Guo Y, Yang X, Zhang X, Wu Y, Yang W, Wang D. Kir2.1-mediated membrane potential promotes nutrient acquisition and inflammation through regulation of nutrient transporters. Nat Commun 2022; 13:3544. [PMID: 35729093 PMCID: PMC9213538 DOI: 10.1038/s41467-022-31149-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Accepted: 05/26/2022] [Indexed: 12/22/2022] Open
Abstract
Immunometabolism contributes to inflammation, but how activated macrophages acquire extracellular nutrients to fuel inflammation is largely unknown. Here, we show that the plasma membrane potential (Vm) of macrophages mediated by Kir2.1, an inwardly-rectifying K+ channel, is an important determinant of nutrient acquisition and subsequent metabolic reprogramming promoting inflammation. In the absence of Kir2.1 activity, depolarized macrophage Vm lead to a caloric restriction state by limiting nutrient uptake and concomitant adaptations in nutrient conservation inducing autophagy, AMPK (Adenosine 5'-monophosphate-activated protein kinase), and GCN2 (General control nonderepressible 2), which subsequently depletes epigenetic substrates feeding histone methylation at loci of a cluster of metabolism-responsive inflammatory genes, thereby suppressing their transcription. Kir2.1-mediated Vm supports nutrient uptake by facilitating cell-surface retention of nutrient transporters such as 4F2hc and GLUT1 by its modulation of plasma membrane phospholipid dynamics. Pharmacological targeting of Kir2.1 alleviated inflammation triggered by LPS or bacterial infection in a sepsis model and sterile inflammation in human samples. These findings identify an ionic control of macrophage activation and advance our understanding of the immunomodulatory properties of Vm that links nutrient inputs to inflammatory diseases.
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Affiliation(s)
- Weiwei Yu
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, P. R. China
| | - Zhen Wang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China.,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, P. R. China
| | - Xiafei Yu
- Department of Biophysics, and Department of Neurology of the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Yonghui Zhao
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China
| | - Zili Xie
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China
| | - Kailian Zhang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Zhexu Chi
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Sheng Chen
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Ting Xu
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Danlu Jiang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Xingchen Guo
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China
| | - Mobai Li
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Jian Zhang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Hui Fang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Dehang Yang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Yuxian Guo
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Xuyan Yang
- Department of Rheumatology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Xue Zhang
- Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China
| | - Yingliang Wu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China
| | - Wei Yang
- Department of Biophysics, and Department of Neurology of the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China.
| | - Di Wang
- Institute of Immunology and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, P. R. China. .,Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou, P. R. China.
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13
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Han J, Zhang H, Li N, Aziz AUR, Zhang Z, Liu B. The raft cytoskeleton binding protein complexes personate functional regulators in cell behaviors. Acta Histochem 2022; 124:151859. [PMID: 35123353 DOI: 10.1016/j.acthis.2022.151859] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Revised: 01/23/2022] [Accepted: 01/27/2022] [Indexed: 12/08/2022]
Abstract
Several cytoskeleton proteins interact with raft proteins to form raft-cytoskeleton binding protein complexes (RCPCs) that control cell migration and adhesion. The purpose of this paper is to review the latest research on the modes and mechanisms by which a RCPC controls different cellular functions. This paper discusses RCPC composition and its role in cytoskeleton reorganization, as well as the latest developments in molecular mechanisms that regulate cell adhesion and migration under normal conditions. In addition, the role of some external stimuli (such as stress and chemical signals) in this process is further debated, and meanwhile potential mechanisms for RCPC to regulate lipid raft fluidity is proposed. Thus, this review mainly contributes to the understanding of RCPC signal transduction in cells. Additionally, the targeted signal transduction of RCPC and its mechanism connection with cell behaviors will provide a logical basis for the development of unified interventions to combat metastasis related dysfunction and diseases.
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Affiliation(s)
- Jinxin Han
- School of Biomedical Engineering, Dalian University of Technology, Key Laboratory for Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian 116024, China
| | - Hangyu Zhang
- School of Biomedical Engineering, Dalian University of Technology, Key Laboratory for Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian 116024, China
| | - Na Li
- School of Biomedical Engineering, Dalian University of Technology, Key Laboratory for Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian 116024, China
| | - Aziz Ur Rehman Aziz
- School of Biomedical Engineering, Dalian University of Technology, Key Laboratory for Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian 116024, China
| | - Zhengyao Zhang
- School of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin 124221, China.
| | - Bo Liu
- School of Biomedical Engineering, Dalian University of Technology, Key Laboratory for Integrated Circuit and Biomedical Electronic System of Liaoning Province, Dalian 116024, China.
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14
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Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles. Int J Mol Sci 2021; 22:ijms222312960. [PMID: 34884763 PMCID: PMC8657571 DOI: 10.3390/ijms222312960] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 11/25/2021] [Accepted: 11/25/2021] [Indexed: 11/17/2022] Open
Abstract
The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.
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15
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Kokhanyuk B, Bodó K, Sétáló G, Németh P, Engelmann P. Bacterial Engulfment Mechanism Is Strongly Conserved in Evolution Between Earthworm and Human Immune Cells. Front Immunol 2021; 12:733541. [PMID: 34539669 PMCID: PMC8440998 DOI: 10.3389/fimmu.2021.733541] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 08/12/2021] [Indexed: 12/13/2022] Open
Abstract
Invertebrates, including earthworms, are applied to study the evolutionarily conserved cellular immune processes. Earthworm immunocytes (so-called coelomocytes) are functionally similar to vertebrate myeloid cells and form the first line of defense against invading pathogens. Hereby, we compared the engulfment mechanisms of THP-1 human monocytic cells, differentiated THP-1 (macrophage-like) cells, and Eisenia andrei coelomocytes towards Escherichia coli and Staphylococcus aureus bacteria applying various endocytosis inhibitors [amantadine, 5-(N-ethyl-N-isopropyl) amiloride, colchicine, cytochalasin B, cytochalasin D, methyl-ß-cyclodextrin, and nystatin]. Subsequently, we investigated the messenger RNA (mRNA) expressions of immune receptor-related molecules (TLR, MyD88, BPI) and the colocalization of lysosomes with engulfed bacteria following uptake inhibition in every cell type. Actin depolymerization by cytochalasin B and D has strongly inhibited the endocytosis of both bacterial strains in the studied cell types, suggesting the conserved role of actin-dependent phagocytosis. Decreased numbers of colocalized lysosomes/bacteria supported these findings. In THP-1 cells TLR expression was increased upon cytochalasin D pretreatment, while this inhibitor caused a dropped LBP/BPI expression in differentiated THP-1 cells and coelomocytes. The obtained data reveal further insights into the evolution of phagocytes in eukaryotes. Earthworm and human phagocytes possess analogous mechanisms for bacterial internalization.
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Affiliation(s)
- Bohdana Kokhanyuk
- Department of Immunology and Biotechnology, Clinical Center, Medical School, University of Pécs, Pécs, Hungary
| | - Kornélia Bodó
- Department of Immunology and Biotechnology, Clinical Center, Medical School, University of Pécs, Pécs, Hungary
| | - György Sétáló
- Department of Medical Biology and Central Electron Microscope Laboratory, Medical School, University of Pécs, Pécs, Hungary.,Signal Transduction Research Group, János Szentágothai Research Centre, Pécs, Hungary
| | - Péter Németh
- Department of Immunology and Biotechnology, Clinical Center, Medical School, University of Pécs, Pécs, Hungary
| | - Péter Engelmann
- Department of Immunology and Biotechnology, Clinical Center, Medical School, University of Pécs, Pécs, Hungary
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16
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Capera J, Pérez-Verdaguer M, Navarro-Pérez M, Felipe A. Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression. Cancers (Basel) 2021; 13:cancers13174457. [PMID: 34503267 PMCID: PMC8431373 DOI: 10.3390/cancers13174457] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 08/27/2021] [Accepted: 09/03/2021] [Indexed: 12/12/2022] Open
Abstract
Simple Summary Voltage-dependent potassium channels control the proliferation of mammalian cells. In addition, mitochondria physiology is highly dynamic during the cell cycle. The aim of this work was to investigate whether the Kv1.3 channel participates in the mitochondrial control of cell cycle progression. Our data confirmed that Kv1.3 facilitates the proliferation of preadipocytes through the control of mitochondrial dynamics. In addition, adipogenesis was also dependent on Kv1.3 expression. We shed light on the role of Kv1.3 in mitochondria and adipose tissue metabolism, contributing further to the control of cell proliferation by Kv1.3. Abstract The voltage-gated potassium channel Kv1.3 is a potential therapeutic target for obesity and diabetes. The genetic ablation and pharmacological inhibition of Kv1.3 lead to a lean phenotype in rodents. The mechanism of regulation of body weight and energy homeostasis involves Kv1.3 expression in different organs, including white and brown adipose tissues. Here, we show that Kv1.3 promotes the proliferation of preadipocytes through the control of mitochondrial dynamics. Kv1.3 is expressed in mitochondria exhibiting high affinity for the perinuclear population. The mitochondrial network is highly dynamic during the cell cycle, showing continuous fusion-fission events. The formation of a hyperfused mitochondrial network at the G1/S phase of the cell cycle is dependent on Kv1.3 expression. Our results demonstrate that Kv1.3 promotes preadipocyte proliferation and differentiation by controlling mitochondrial membrane potential and mitochondrial dynamics at the G1 phase of the cell cycle.
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Affiliation(s)
- Jesusa Capera
- Molecular Physiology Laboratory, Departament de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain; (J.C.); (M.P.-V.); (M.N.-P.)
- Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, UK
| | - Mireia Pérez-Verdaguer
- Molecular Physiology Laboratory, Departament de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain; (J.C.); (M.P.-V.); (M.N.-P.)
- Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - María Navarro-Pérez
- Molecular Physiology Laboratory, Departament de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain; (J.C.); (M.P.-V.); (M.N.-P.)
| | - Antonio Felipe
- Molecular Physiology Laboratory, Departament de Bioquímica i Biomedicina Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain; (J.C.); (M.P.-V.); (M.N.-P.)
- Correspondence:
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17
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Yılmaz D, Culha M. Investigation of the pathway dependent endocytosis of gold nanoparticles by surface-enhanced Raman scattering. Talanta 2021; 225:122071. [PMID: 33592789 DOI: 10.1016/j.talanta.2020.122071] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Revised: 12/15/2020] [Accepted: 12/27/2020] [Indexed: 01/02/2023]
Abstract
Endocytosis is a critical mechanism providing not only internalization of biomacromolecular structures but also communication with the environment where cells reside. Due to being the first step at the interaction interface, the route of cellular uptake has a major role governing the intracellular destinations and behaviors of molecular and non-molecular species including nanoparticles. To this end, various methods employing variety of techniques are investigated. In this study, surface-enhanced Raman spectroscopy (SERS) based approach for the investigation of endocytosis of gold nanoparticles (AuNPs) is reported. Internalization pathways of AuNPs were examined by flow cytometry via specific inhibitors for each endocytosis pathway type using three model cell lines Beas-2b, A549 and PNT1A. Macropinocytosis was blocked by cytochalasin D (CytoD), clathrin mediated endocytosis (CME) by sucrose (Scr), and caveolae mediated endocytosis (CE) by filipin (Fil). The results showed that cell type dependent AuNPs internalization affects not only the response of the cells to the inhibitors but also the obtained SERS spectra. SERS spectra of PNT1A cells treated with inhibitors was influenced most. The inhibition of each endocytosis pathway significantly affected the SERS spectral pattern and the spectral changes in different endocytosis pathways were clearly discriminated from each other. This means that SERS can significantly contribute to the investigation of different endosomal pathways from single living cells without any disruption of the cells or labeling.
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Affiliation(s)
- Deniz Yılmaz
- Yeditepe University, Faculty of Engineering, Department of Genetics and Bioengineering, 34755, Istanbul, Turkey
| | - Mustafa Culha
- Yeditepe University, Faculty of Engineering, Department of Genetics and Bioengineering, 34755, Istanbul, Turkey; Oregon Health and Science University, The Knight Cancer Research Institute, Cancer Early Detection Advanced Research (CEDAR) Center, Portland, OR 97239 USA; Sabanci University Nanotechnology Research and Application Center (SUNUM), Tuzla, Istanbul, 34956 Turkey.
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18
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Pinton L, Magri S, Masetto E, Vettore M, Schibuola I, Ingangi V, Marigo I, Matha K, Benoit JP, Della Puppa A, Bronte V, Lollo G, Mandruzzato S. Targeting of immunosuppressive myeloid cells from glioblastoma patients by modulation of size and surface charge of lipid nanocapsules. J Nanobiotechnology 2020; 18:31. [PMID: 32066449 PMCID: PMC7026969 DOI: 10.1186/s12951-020-00589-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Accepted: 01/30/2020] [Indexed: 12/11/2022] Open
Abstract
Background Myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are two of the major players involved in the inhibition of anti-tumor immune response in cancer patients, leading to poor prognosis. Selective targeting of myeloid cells has therefore become an attractive therapeutic strategy to relieve immunosuppression and, in this frame, we previously demonstrated that lipid nanocapsules (LNCs) loaded with lauroyl-modified gemcitabine efficiently target monocytic MDSCs in melanoma patients. In this study, we investigated the impact of the physico-chemical characteristics of LNCs, namely size and surface potential, towards immunosuppressive cell targeting. We exploited myeloid cells isolated from glioblastoma patients, which play a relevant role in the immunosuppression, to demonstrate that tailored nanosystems can target not only tumor cells but also tumor-promoting cells, thus constituting an efficient system that could be used to inhibit their function. Results The incorporation of different LNC formulations with a size of 100 nm, carrying overall positive, neutral or negative charge, was evaluated on leukocytes and tumor-infiltrating cells freshly isolated from glioblastoma patients. We observed that the maximum LNC uptake was obtained in monocytes with neutral 100 nm LNCs, while positively charged 100 nm LNCs were more effective on macrophages and tumor cells, maintaining at low level the incorporation by T cells. The mechanism of uptake was elucidated, demonstrating that LNCs are incorporated mainly by caveolae-mediated endocytosis. Conclusions We demonstrated that LNCs can be directed towards immunosuppressive cells by simply modulating their size and charge thus providing a novel approach to exploit nanosystems for anticancer treatment in the frame of immunotherapy.![]()
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Affiliation(s)
- Laura Pinton
- Veneto Institute of Oncology IOV-IRCCS, Padua, Italy
| | - Sara Magri
- Department of Surgery, Oncology and Gastroenterology, University of Padova, Via Gattamelata 64, 35128, Padua, Italy
| | - Elena Masetto
- Veneto Institute of Oncology IOV-IRCCS, Padua, Italy
| | | | - Ilaria Schibuola
- Department of Surgery, Oncology and Gastroenterology, University of Padova, Via Gattamelata 64, 35128, Padua, Italy
| | | | - Ilaria Marigo
- Veneto Institute of Oncology IOV-IRCCS, Padua, Italy
| | - Kevin Matha
- Pharmacy Department, Academic Hospital, 4 rue Larrey, Angers, France.,Micro et Nanomedecines Translationnelles, MINT, UNIV Angers, UMR INSERM 1066, UMR CNRS 6021, Angers, France
| | - Jean-Pierre Benoit
- Pharmacy Department, Academic Hospital, 4 rue Larrey, Angers, France.,Micro et Nanomedecines Translationnelles, MINT, UNIV Angers, UMR INSERM 1066, UMR CNRS 6021, Angers, France
| | - Alessandro Della Puppa
- Neurosurgery Unit, Azienda Ospedaliera di Padova, Padua, Italy.,Department of NEUROFARBA, University Hospital of Careggi, University of Florence, Florence, Italy
| | - Vincenzo Bronte
- Department of Medicine, Section of Immunology, University of Verona, Verona, Italy
| | - Giovanna Lollo
- Univ Lyon, Université Claude Bernard Lyon 1, CNRS, LAGEPP UMR 5007, 69100, Villeurbanne, France
| | - Susanna Mandruzzato
- Veneto Institute of Oncology IOV-IRCCS, Padua, Italy. .,Department of Surgery, Oncology and Gastroenterology, University of Padova, Via Gattamelata 64, 35128, Padua, Italy.
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19
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Fong VH, Wong S, Jintaridhi P, Vieira A. Transport of the Thyroid Hormone Carrier Protein Transthyretin into Human Epidermoid Cells. Endocr Res 2020; 45:131-136. [PMID: 31762320 DOI: 10.1080/07435800.2019.1694538] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Purpose: Transthyretin (TTR) is a protein with a growing number of biological functions in addition to its well-established binding and circulatory transport of thyroxine, and indirect retinoid transport through interaction with retinol-binding protein. Misfolded and aggregated wild-type and mutant TTRs are involved in amyloid diseases. Several aspects of TTR pathology and physiology remain poorly understood. Receptor-mediated cellular transport of TTR has been described in a few cell types; and such studies suggest the possibility of different TTR receptors and endocytic pathways. Our main objective was to further understand the endocytic pathways for TTR.Methods: In the current study, analyses of TTR endocytic transport were performed in the human A431 cell line. The results of TTR uptake were compared with those of the iron-carrier protein transferrin (Tf, a common stardard for endocytosis studies) in the same cell types.Results: A comparison of TTR and Tf endocytosis suggested similar early, 5-10 min, accumulation kinetics. But at a later time, 30 min, TTR accumulation was 20-30% lower than that of Tf (p < .05), a result that suggests different post-endocytic fates for these two ligands. Through the use of multiple endocytosis inhibitors, biochemical evidence is provided for an internalization pathway that differs from the clathrin-mediated endocytosis of Tf.Conclusions: These results for A431 cells are compared with others reported for different cell types; and it is suggested that this same hormone carrier protein can transit into cells through multiple endocytic pathways.
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Affiliation(s)
- Vai Hong Fong
- Biomedical Physiology BPK, Simon Fraser University, Burnaby, BC, Canada
- Department of Neurology, Far Eastern Memorial University Hospital, New Taipei City, Taiwan
| | - Shaun Wong
- Biomedical Physiology BPK, Simon Fraser University, Burnaby, BC, Canada
| | | | - Amandio Vieira
- Biomedical Physiology BPK, Simon Fraser University, Burnaby, BC, Canada
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20
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Abstract
Selectively targeting the cell nucleolus remains a challenge. Here, we report the first case in which d-peptides form membraneless molecular condensates with RNA for targeting cell nucleolus. A d-peptide derivative, enriched with lysine and hydrophobic residues, self-assembles to form nanoparticles, which enter cells through clathrin-dependent endocytosis and mainly accumulate at the cell nucleolus. A structural analogue of the d-peptide reveals that the particle morphology of the assemblies, which depends on the side chain modification, favors the cellular uptake. In contrast to those of the d-peptide, the assemblies of the corresponding l-enantiomer largely localize in cell lysosomes. Preliminary mechanism study suggests that the d-peptide nanoparticles interact with RNA to form membraneless condensates in the nucleolus, which further induces DNA damage and results in cell death. This work illustrates a new strategy for rationally designing supramolecular assemblies of d-peptides for targeting subcellular organelles.
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Affiliation(s)
- Huaimin Wang
- Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA
| | - Zhaoqianqi Feng
- Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA
| | - Weiyi Tan
- Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA
| | - Bing Xu
- Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02454, USA
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21
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Chiu PC, Hsieh PY, Kang JW, Chang PH, Shen LJ. Study of the intracellular delivery mechanism of a pH-sensitive peptide modified with enhanced green fluorescent protein. J Drug Target 2019; 28:408-418. [PMID: 31524004 DOI: 10.1080/1061186x.2019.1669041] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The targeted delivery of therapeutic agents is a promising approach to enhance the efficacy and reduce the toxicity of cancer treatments. Understanding the intracellular endocytic mechanisms of a cell penetrating peptide (CPP) in an acidic environment is important for targeted delivery of macromolecules to tumours. In this study, we constructed a pH-sensitive CPP-based delivery system for the intracellular delivery of macromolecules. A pH-sensitive CPP, HBHAc, was fused with a model protein, enhanced green fluorescent protein (EGFP), through recombinant DNA technology. We found that is essential that negatively charged proteoglycans on the cell surface interact with HBHAc-EGFP prior to the cellular uptake of HBHAc-EGFP. The uptake was significantly restricted at 4 °C under pH conditions of both 6.5 and 7.5. The increased positive charge of HBHAc-EGFP under the acidic condition leads to a pH-dependent cellular uptake, and we observed that the internalisation of HBHAc-EGFP was significantly higher at pH 6.5 than at pH 7.5 (p < .05). Thus, with pH-sensitive activity, HBHAc is expected to improve tumour-targeted intracellular protein delivery. Moreover, our findings provide a new insight that the endocytic pathway may change under different pH conditions and suggest that this unique phenomenon benefits pH-sensitive drug delivery for tumour therapy.
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Affiliation(s)
- Po-Chuan Chiu
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Pei-Yu Hsieh
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Jyun-Wei Kang
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Po-Hsun Chang
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Li-Jiuan Shen
- School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.,Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.,Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan
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22
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Antinozzi C, Marampon F, Sgrò P, Tombolini V, Lenzi A, Crescioli C, Di Luigi L. Comparative study of testosterone and vitamin D analogue, elocalcitol, on insulin-controlled signal transduction pathway regulation in human skeletal muscle cells. J Endocrinol Invest 2019; 42:897-907. [PMID: 30600434 DOI: 10.1007/s40618-018-0998-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2018] [Accepted: 12/21/2018] [Indexed: 12/19/2022]
Abstract
PURPOSE Skeletal muscle (Skm) plays a key role in regulating energetic metabolism through glucose homeostasis. Several hormones such as Testosterone (T) and Vitamin D (VD) have been shown to affect energy-dependent cell trafficking by determining Insulin (I)-like effects. AIM To elucidate possible hormone-related differences on muscular metabolic control, we analyzed and compared the effects of T and elocalcitol (elo), a VD analogue, on the activation of energy-dependent cell trafficking, metabolism-related-signal transduction pathways and transcription of gene downstream targets. METHODS Human fetal skeletal muscle cells (Hfsmc) treated with T or elo were analyzed for GLUT4 localization, phosphorylation/activation status of AKT, ERK1/2, IRS-1 signaling and c-MYC protein expression. RESULTS T, similar to elo, induced GLUT4 protein translocation likely in lipid raft microdomains. While both T and elo induced a rapid IRS-1 phosphorylation, the following dynamic in phosphorylation/activation of AKT and ERK1/2 signaling was different. Moreover, T but not elo increased c-MYC protein expression. CONCLUSIONS All together, our evidence indicates that whether both T and elo are able to affect upstream I-like pathway, they differently determine downstream effects in I-dependent cascade, suggesting diverse physiological roles in mediating I-like response in human skeletal muscle.
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Affiliation(s)
- C Antinozzi
- Unit of Endocrinology, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135, Rome, Italy
| | - F Marampon
- Unit of Endocrinology, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135, Rome, Italy
- Department of Radiotherapy, Sapienza University of Rome, Rome, Italy
| | - P Sgrò
- Unit of Endocrinology, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135, Rome, Italy
| | - V Tombolini
- Department of Radiotherapy, Sapienza University of Rome, Rome, Italy
| | - A Lenzi
- Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
| | - C Crescioli
- Unit of Endocrinology, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135, Rome, Italy.
| | - L Di Luigi
- Unit of Endocrinology, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135, Rome, Italy
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Song G, Zong C, Shao M, Yu Y, Liu Q, Wang H, Qiu T, Jiao P, Guo Z, Lee P, Luo Y, Jiang XC, Qin S. Phospholipid transfer protein (PLTP) deficiency attenuates high fat diet induced obesity and insulin resistance. Biochim Biophys Acta Mol Cell Biol Lipids 2019; 1864:1305-1313. [PMID: 31220615 DOI: 10.1016/j.bbalip.2019.06.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 06/07/2019] [Accepted: 06/14/2019] [Indexed: 01/09/2023]
Abstract
Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the PLTP deficiency induced significantly more GLUT4 protein in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP-deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increase in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulating lipid levels in the plasma membrane, especially in the lipid rafts.
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Affiliation(s)
- Guohua Song
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China.
| | - Chuanlong Zong
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Mingzhu Shao
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Yang Yu
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Qian Liu
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Hui Wang
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Tingting Qiu
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Peng Jiao
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Zheng Guo
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China
| | - Phoebe Lee
- Downstate Medical Center State University of New York, NY, USA
| | - Yi Luo
- Downstate Medical Center State University of New York, NY, USA
| | | | - Shucun Qin
- Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, China.
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Sabharwal P, Amritha CK, Sushmitha C, Natraj U, Savithri HS. Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells. Nanomedicine (Lond) 2019; 14:1247-1265. [PMID: 31084385 DOI: 10.2217/nnm-2018-0405] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.
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Affiliation(s)
- Pallavi Sabharwal
- Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
| | | | - Cheekati Sushmitha
- Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
| | - Usha Natraj
- Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
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Pérez-Verdaguer M, Capera J, Ortego-Domínguez M, Bielanska J, Comes N, Montoro RJ, Camps M, Felipe A. Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology. Cell Mol Life Sci 2018; 75:4059-4075. [PMID: 29947924 PMCID: PMC11105548 DOI: 10.1007/s00018-018-2851-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 05/14/2018] [Accepted: 06/05/2018] [Indexed: 12/23/2022]
Abstract
The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.
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Affiliation(s)
- Mireia Pérez-Verdaguer
- Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
- Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
| | - Jesusa Capera
- Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
- Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
| | - María Ortego-Domínguez
- Dpto. de Fisiología Médica y Biofísica, Universidad de Sevilla, Av. Dr. Fedriani, s/n., 41009, Seville, Spain
| | - Joanna Bielanska
- Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
- Max-Planck-Institute of Experimental Medicine, Molecular Biology of Neuronal Signals, AG Oncophysiology, Hermann-Rein-Str. 3, 37075, Göttingen, Germany
| | - Núria Comes
- Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
| | - Rafael J Montoro
- Dpto. de Fisiología Médica y Biofísica, Universidad de Sevilla, Av. Dr. Fedriani, s/n., 41009, Seville, Spain
| | - Marta Camps
- Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain
| | - Antonio Felipe
- Molecular Physiology Laboratory, Dpt. de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
- Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
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Xue Z, Wang S, Li J, Chen X, Han J, Han S. Bifunctional Super-resolution Imaging Probe with Acidity-Independent Lysosome-Retention Mechanism. Anal Chem 2018; 90:11393-11400. [DOI: 10.1021/acs.analchem.8b02365] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Zhongwei Xue
- Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University, Xiamen, 361005, China
| | - Siyu Wang
- Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University, Xiamen, 361005, China
| | - Jian Li
- Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University, Xiamen, 361005, China
| | - Xin Chen
- State key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China
| | - Jiahuai Han
- State key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China
| | - Shoufa Han
- Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University, Xiamen, 361005, China
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27
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Ishii T, Miyauchi K, Nitta Y, Kaneko K, Maruyama T, Sato T. Mechanism for Decreased Gene Expression of β4-Galactosyltransferase 5 upon Differentiation of 3T3-L1 Mouse Preadipocytes to Adipocytes. Biol Pharm Bull 2018; 41:1463-1470. [PMID: 29984736 DOI: 10.1248/bpb.b18-00360] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Upon differentiation of cells, remarkable changes in the structures of glycans linked to lipids on cell surface have been observed. Lactosylceramide (Lac-Cer) serves as a common precursor for a series of glycosphingolipids with diverse structures. In the present study, we examined the underlying mechanism for the biosynthesis of Lac-Cer upon differentiation of 3T3-L1 mouse preadipocytes to adipocytes. TLC analysis showed that the amounts of Lac-Cer decrease in 3T3-L1 adipocytes compared to 3T3-L1 preadipocytes. In accordance with this change, the gene expression level of β4-galactosyltransferase (β4GalT) 5, which was identified as Lac-Cer synthase, decreased drastically upon differentiation of 3T3-L1 preadipocytes. The analysis of the transcriptional mechanism of the β4GalT5 gene demonstrated that the core promoter region is identified between nucleotides -299 and -1 relative to the translational start site. During adipocyte differentiation, the expression levels and promoter activities of the β4GalT5 gene decreased dramatically. Since the Specificity protein 1 (Sp1)-binding sites in the promoter region were critical for the promoter activity, it is suggested that Sp1 plays an important role for the expression of the β4GalT5 gene in 3T3-L1 cells. The gene and protein expression of Sp1 decreased significantly upon differentiation of 3T3-L1 preadipocytes. Taken together, the present study suggest that the expression of the β4GalT5 gene decreases through reduced expression of the Sp1 gene and protein upon differentiation of 3T3-L1 peradipocytes to adipocytes, which may lead to the decreased amounts of Lac-Cer in 3T3-L1 adipocytes.
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Affiliation(s)
- Takayuki Ishii
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
| | - Kana Miyauchi
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
| | - Yoshiharu Nitta
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
| | - Kazuhiro Kaneko
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
| | - Takuro Maruyama
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
| | - Takeshi Sato
- Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology
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28
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Guo N, Gao C, Liu J, Li J, Liu N, Hao Y, Chen L, Zhang X. Reversal of Ovarian Cancer Multidrug Resistance by a Combination of LAH4-L1-siMDR1 Nanocomplexes with Chemotherapeutics. Mol Pharm 2018; 15:1853-1861. [PMID: 29621396 DOI: 10.1021/acs.molpharmaceut.8b00031] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Nana Guo
- Collaborative Innovation Center for Biotherapy, Tsinghua University, Beijing 100084, China
- Anhui Medical University, Hefei, Anhui 230032, China
- Department of Gynaecology and Obstetrics, PLA Navy General Hospital, Beijing 100037, China
| | - Chen Gao
- College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China
| | - Jing Liu
- Collaborative Innovation Center for Biotherapy, Tsinghua University, Beijing 100084, China
| | - Jun Li
- School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China
| | - Nan Liu
- School of Medicine, Tsinghua University, Beijing 100084, China
| | - Yanli Hao
- School of Medicine, Tsinghua University, Beijing 100084, China
| | - Lei Chen
- Anhui Medical University, Hefei, Anhui 230032, China
- Department of Gynaecology and Obstetrics, PLA Navy General Hospital, Beijing 100037, China
| | - Xiaoning Zhang
- Collaborative Innovation Center for Biotherapy, Tsinghua University, Beijing 100084, China
- School of Medicine, Tsinghua University, Beijing 100084, China
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29
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Marampon F, Antinozzi C, Corinaldesi C, Vannelli GB, Sarchielli E, Migliaccio S, Di Luigi L, Lenzi A, Crescioli C. The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism. Endocrine 2018; 59:602-613. [PMID: 28786077 DOI: 10.1007/s12020-017-1378-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/11/2017] [Accepted: 07/20/2017] [Indexed: 01/01/2023]
Abstract
PURPOSE Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. METHODS RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. RESULTS Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. CONCLUSION In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.
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Affiliation(s)
- F Marampon
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy
| | - C Antinozzi
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy
| | - C Corinaldesi
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK
| | - G B Vannelli
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - E Sarchielli
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - S Migliaccio
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy
| | - L Di Luigi
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy
| | - A Lenzi
- Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy
| | - C Crescioli
- Department of Movement, Human and Health Sciences, Università di Roma "Foro Italico", Rome, Italy.
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Zhang X, Ren J, Wang J, Li S, Zou Q, Gao N. Receptor-mediated endocytosis generates nanomechanical force reflective of ligand identity and cellular property. J Cell Physiol 2018; 233:5908-5919. [PMID: 29243828 DOI: 10.1002/jcp.26400] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 12/12/2017] [Indexed: 01/07/2023]
Abstract
Whether environmental (thermal, chemical, and nutrient) signals generate quantifiable, nanoscale, mechanophysical changes in the cellular plasma membrane has not been well elucidated. Assessment of such mechanophysical properties of plasma membrane may shed lights on fundamental cellular process. Atomic force microscopic (AFM) measurement of the mechanical properties of live cells was hampered by the difficulty in accounting for the effects of the cantilever motion and the associated hydrodynamic force on the mechanical measurement. These challenges have been addressed in our recently developed control-based AFM nanomechanical measurement protocol, which enables a fast, noninvasive, broadband measurement of the real-time changes in plasma membrane elasticity in live cells. Here we show using this newly developed AFM platform that the plasma membrane of live mammalian cells exhibits a constant and quantifiable nanomechanical property, the membrane elasticity. This mechanical property sensitively changes in response to environmental factors, such as the thermal, chemical, and growth factor stimuli. We demonstrate that different chemical inhibitors of endocytosis elicit distinct changes in plasma membrane elastic modulus reflecting their specific molecular actions on the lipid configuration or the endocytic machinery. Interestingly, two different growth factors, EGF and Wnt3a, elicited distinct elastic force profiles revealed by AFM at the plasma membrane during receptor-mediated endocytosis. By applying this platform to genetically modified cells, we uncovered a previously unknown contribution of Cdc42, a key component of the cellular trafficking network, to EGF-stimulated endocytosis at plasma membrane. Together, this nanomechanical AFM study establishes an important foundation that is expandable and adaptable for investigation of cellular membrane evolution in response to various key extracellular signals.
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Affiliation(s)
- Xiao Zhang
- Department of Biological Sciences, Rutgers University, Newark, New Jersey
| | - Juan Ren
- Department of Mechanical Engineering, Iowa State University, Ames, Iowa
| | - Jingren Wang
- Department of Mechanical and Aerospace Engineering, Rutgers University, Piscataway, New Jersey
| | - Shixie Li
- Department of Mechanical and Aerospace Engineering, Rutgers University, Piscataway, New Jersey
| | - Qingze Zou
- Department of Mechanical and Aerospace Engineering, Rutgers University, Piscataway, New Jersey
| | - Nan Gao
- Department of Biological Sciences, Rutgers University, Newark, New Jersey
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Sun B, Zhong Z, Wang F, Xu J, Xu F, Kong W, Ling Z, Shu N, Li Y, Wu T, Zhang M, Zhu L, Liu X, Liu L. Atorvastatin impaired glucose metabolism in C2C12 cells partly via inhibiting cholesterol-dependent glucose transporter 4 translocation. Biochem Pharmacol 2018; 150:108-119. [PMID: 29338971 DOI: 10.1016/j.bcp.2018.01.021] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 01/08/2018] [Indexed: 10/18/2022]
Abstract
Skeletal muscle accounts for approximately 75% of glucose disposal in body and statins impair glucose metabolism. We aimed to investigate the effect of atorvastatin on glucose metabolism in C2C12 cells. Glucose metabolism and expression of glucose transporter 4 (GLUT4) and hexokinase II (HXKII) were measured following incubation with atorvastatin or pravastatin. Roles of cholesterol in atorvastatin-induced glucose metabolism impairment were investigated via adding cholesterol or mevalonic acid and confirmed by cholesterol depletion with methyl-β-cyclodextrin. Hypercholesterolemia mice induced by high fat diet (HFD) feeding, orally received atorvastatin (6 and 12 mg/kg) or pravastatin (12 mg/kg) for 22 days. Results showed that atorvastatin not pravastatin concentration-dependently impaired glucose consumption, glucose uptake and GLUT4 membrane translocation in C2C12 cells without affecting expression of HXKII or total GLUT4 protein. The atorvastatin-induced alterations were reversed by cholesterol or mevalonic acid. Cholesterol depletion exerted similar impact to atorvastatin, which could be alleviated by cholesterol supplement. Glucose consumption or GLUT4 translocation was positively associated with cellular cholesterol levels. In HFD mice, atorvastatin not pravastatin significantly increased blood glucose levels following glucose or insulin dose and decreased expression of membrane not total GLUT4 protein in muscle. Glucose exposure following glucose or insulin dose was negatively correlated to muscular free cholesterol concentration. Expression of membrane GLUT4 protein was positively related to free cholesterol in muscle. In conclusion, atorvastatin impaired glucose utilization in muscle cells partly via inhibiting GLUT4 membrane translocation due to inhibition of cholesterol synthesis by atorvastatin, at least, partly contributing to glucose intolerance in HFD mice.
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Affiliation(s)
- Binbin Sun
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Zeyu Zhong
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Fan Wang
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Jiong Xu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Feng Xu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Weimin Kong
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Zhaoli Ling
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Nan Shu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Ying Li
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Tong Wu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Mian Zhang
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Liang Zhu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Xiaodong Liu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
| | - Li Liu
- Center of Drug Metabolism and Pharmacokinetics, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
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Harwardt MLIE, Young P, Bleymüller WM, Meyer T, Karathanasis C, Niemann HH, Heilemann M, Dietz MS. Membrane dynamics of resting and internalin B-bound MET receptor tyrosine kinase studied by single-molecule tracking. FEBS Open Bio 2017; 7:1422-1440. [PMID: 28904870 PMCID: PMC5586345 DOI: 10.1002/2211-5463.12285] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Revised: 08/07/2017] [Accepted: 08/08/2017] [Indexed: 11/09/2022] Open
Abstract
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET. We show that internalin B-bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B-bound receptors than for resting MET. Results of single-particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.
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Affiliation(s)
- Marie-Lena I E Harwardt
- Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt Germany
| | - Phoebe Young
- Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt Germany
| | - Willem M Bleymüller
- Structural Biochemistry Department of Chemistry Bielefeld University Germany
| | - Timo Meyer
- Structural Biochemistry Department of Chemistry Bielefeld University Germany
| | - Christos Karathanasis
- Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt Germany
| | - Hartmut H Niemann
- Structural Biochemistry Department of Chemistry Bielefeld University Germany
| | - Mike Heilemann
- Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt Germany
| | - Marina S Dietz
- Institute of Physical and Theoretical Chemistry Johann Wolfgang Goethe-University Frankfurt Germany
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33
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Desai AJ, Miller LJ. Changes in the plasma membrane in metabolic disease: impact of the membrane environment on G protein-coupled receptor structure and function. Br J Pharmacol 2017; 175:4009-4025. [PMID: 28691227 DOI: 10.1111/bph.13943] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Revised: 06/08/2017] [Accepted: 07/04/2017] [Indexed: 12/11/2022] Open
Abstract
Drug development targeting GPCRs often utilizes model heterologous cell expression systems, reflecting an implicit assumption that the membrane environment has little functional impact on these receptors or on their responsiveness to drugs. However, much recent data have illustrated that membrane components can have an important functional impact on intrinsic membrane proteins. This review is directed toward gaining a better understanding of the structure of the plasma membrane in health and disease, and how this organelle can influence GPCR structure, function and regulation. It is important to recognize that the membrane provides a potential mode of lateral allosteric regulation of GPCRs and can affect the effectiveness of drugs and their biological responses in various disease states, which can even vary among individuals across the population. The type 1 cholecystokinin receptor is reviewed as an exemplar of a class A GPCR that is affected in this way by changes in the plasma membrane. LINKED ARTICLES This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.
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Affiliation(s)
- Aditya J Desai
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA
| | - Laurence J Miller
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA
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34
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Honsho M, Fujiki Y. Plasmalogen homeostasis - regulation of plasmalogen biosynthesis and its physiological consequence in mammals. FEBS Lett 2017; 591:2720-2729. [PMID: 28686302 DOI: 10.1002/1873-3468.12743] [Citation(s) in RCA: 76] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2015] [Revised: 06/28/2015] [Accepted: 06/29/2016] [Indexed: 11/06/2022]
Abstract
Plasmalogens, mostly ethanolamine-containing alkenyl ether phospholipids, are a major subclass of glycerophospholipids. Plasmalogen synthesis is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of peroxisome biogenesis-defective patients suggests that the de novo synthesis of plasmalogens plays a pivotal role in its homeostasis in tissues. Plasmalogen synthesis is regulated by modulating the stability of fatty acyl-CoA reductase 1 on peroxisomal membranes, a rate-limiting enzyme in plasmalogen synthesis, by sensing plasmalogens in the inner leaflet of plasma membranes. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis by altering the stability of squalene monooxygenase, a key enzyme in cholesterol biosynthesis, implying physiological consequences of plasmalogen homeostasis with respect to cholesterol metabolism in cells, as well as in organs such as the liver.
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Affiliation(s)
- Masanori Honsho
- Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Yukio Fujiki
- Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
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35
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Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells. J Allergy Clin Immunol 2017; 141:1774-1785.e7. [PMID: 28734845 DOI: 10.1016/j.jaci.2017.06.028] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2016] [Revised: 06/01/2017] [Accepted: 06/12/2017] [Indexed: 01/06/2023]
Abstract
BACKGROUND Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell-surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec-8 surface expression. OBJECTIVES We sought to determine whether Siglec-8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec-8-bearing cells to kill these cells. METHODS Siglec-8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec-8-transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec-8 intracellular trafficking was followed by confocal microscopy. The ribosome-inhibiting protein saporin was conjugated to a Siglec-8-specific antibody to examine the targeting of an agent to these cells through Siglec-8 endocytosis. RESULTS Siglec-8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec-8 localized to the lysosomal compartment. Maximal endocytosis in Siglec-8-transduced HEK293T cells required an intact immunoreceptor tyrosine-based inhibitory motif. Siglec-8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec-8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec-8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC-1.2. CONCLUSIONS Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.
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Plasmalogen biosynthesis is spatiotemporally regulated by sensing plasmalogens in the inner leaflet of plasma membranes. Sci Rep 2017; 7:43936. [PMID: 28272479 PMCID: PMC5341075 DOI: 10.1038/srep43936] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2016] [Accepted: 01/31/2017] [Indexed: 12/14/2022] Open
Abstract
Alkenyl ether phospholipids are a major sub-class of ethanolamine- and choline-phospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. Biosynthesis of ethanolamine-containing alkenyl ether phospholipids, plasmalogens, is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) in a manner dependent on the level of cellular plasmalogens. However, precise molecular mechanisms underlying the regulation of plasmalogen synthesis remain poorly understood. Here we show that degradation of Far1 is accelerated by inhibiting dynamin-, Src kinase-, or flotillin-1-mediated endocytosis without increasing the cellular level of plasmalogens. By contrast, Far1 is stabilized by sequestering cholesterol with nystatin. Moreover, abrogation of the asymmetric distribution of plasmalogens in the plasma membrane by reducing the expression of CDC50A encoding a β-subunit of flippase elevates the expression level of Far1 and plasmalogen synthesis without reducing the total cellular level of plasmalogens. Together, these results support a model that plasmalogens localised in the inner leaflet of the plasma membranes are sensed for plasmalogen homeostasis in cells, thereby suggesting that plasmalogen synthesis is spatiotemporally regulated by monitoring cellular level of plasmalogens.
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Antinozzi C, Corinaldesi C, Giordano C, Pisano A, Cerbelli B, Migliaccio S, Di Luigi L, Stefanantoni K, Vannelli GB, Minisola S, Valesini G, Riccieri V, Lenzi A, Crescioli C. Potential role for the VDR agonist elocalcitol in metabolic control: Evidences in human skeletal muscle cells. J Steroid Biochem Mol Biol 2017; 167:169-181. [PMID: 28042053 DOI: 10.1016/j.jsbmb.2016.12.010] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2016] [Revised: 11/20/2016] [Accepted: 12/20/2016] [Indexed: 12/17/2022]
Abstract
Vitamin D plays a pivotal role to maintain skeletal muscle integrity and health. Vitamin D deficiency characterizes inflammatory myopathy (IM) and diabetes, often overlapping diseases involving skeletal muscle damage. Vitamin D receptor (VDR) agonists likely exert beneficial effects in both IM and metabolic disturbances. We aim to evaluate in vitro the effect of elocalcitol, a non-hypercalcemic VDR agonist, on the biomolecular metabolic machinery of human skeletal muscle cells (Hfsmc), vs. insulin (I). We analyzed GLUT4, Flotillin-1, Caveolin-3 and Caveolin-1 cell expression/localization; mTOR, AKT, ERK and 4E-BP1 phosphorylation; IL-6 myokine release; VDR expression. We investigated in vivo vitamin D status in IM subjects, evaluating VDR muscular expression and serum vitamin D with metabolism-related parameters, as glycemia, triglycerides, cholesterol, resistin and adiponectin. In Hfsmc, elocalcitol exerted an I-like effect, promoting GLUT4 re-localization in Flotillin-1, Caveolin-3 and Caveolin-1 positive sites and mTOR, AKT, ERK, 4E-BP1 activation; it enhanced IL-6 myokine release. IM subjects, all normoglycemic, showed VDR/vitamin D deficiency that, together with high lipidemic and resistin profile, possibly increases the risk to develop metabolic diseases. VDR agonists as elocalcitol may be therapeutic tools for skeletal muscle integrity/function maintenance, an indispensable condition for health homeostasis.
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Affiliation(s)
- Cristina Antinozzi
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, 00135 Rome, Italy
| | - Clarissa Corinaldesi
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, 00135 Rome, Italy; Leeds Institute of Rheumatic & Musculoskeletal Medicine, University of Leeds, Leeds, UK
| | - Carla Giordano
- Department of Radiological, Oncological and Pathological Sciences, Sapienza University of Rome, 00161 Rome, Italy
| | - Annalinda Pisano
- Department of Radiological, Oncological and Pathological Sciences, Sapienza University of Rome, 00161 Rome, Italy
| | - Bruna Cerbelli
- Department of Radiological, Oncological and Pathological Sciences, Sapienza University of Rome, 00161 Rome, Italy
| | - Silvia Migliaccio
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, 00135 Rome, Italy
| | - Luigi Di Luigi
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, 00135 Rome, Italy
| | - Katia Stefanantoni
- Department of Internal Medicine and Medical Specialities, Sapienza University of Rome, 00161 Rome, Italy
| | | | - Salvatore Minisola
- Department of Internal Medicine and Medical Disciplines, Sapienza University of Rome, 00161 Rome, Italy
| | - Guido Valesini
- Department of Internal Medicine and Medical Specialities, Sapienza University of Rome, 00161 Rome, Italy
| | - Valeria Riccieri
- Department of Internal Medicine and Medical Specialities, Sapienza University of Rome, 00161 Rome, Italy
| | - Andrea Lenzi
- Department of Experimental Medicine, Sapienza University of Rome, 00161 Rome, Italy
| | - Clara Crescioli
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, 00135 Rome, Italy.
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Martínez-Mármol R, Styrczewska K, Pérez-Verdaguer M, Vallejo-Gracia A, Comes N, Sorkin A, Felipe A. Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation. Sci Rep 2017; 7:42395. [PMID: 28186199 PMCID: PMC5301257 DOI: 10.1038/srep42395] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 01/09/2017] [Indexed: 12/29/2022] Open
Abstract
The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.
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Affiliation(s)
- Ramón Martínez-Mármol
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain.,Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Katarzyna Styrczewska
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain
| | - Mireia Pérez-Verdaguer
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain
| | - Albert Vallejo-Gracia
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain
| | - Núria Comes
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain.,Laboratory of Neurophysiology, Universitat de Barcelona and Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
| | - Alexander Sorkin
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA
| | - Antonio Felipe
- Molecular Physiology laboratory, Departament de Bioquímica i Biomedicna Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona, 08028 Barcelona, Spain
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Wang H, Feng Z, Wang Y, Zhou R, Yang Z, Xu B. Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance. J Am Chem Soc 2016; 138:16046-16055. [PMID: 27960313 PMCID: PMC5291163 DOI: 10.1021/jacs.6b09783] [Citation(s) in RCA: 209] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.
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Affiliation(s)
- Huaimin Wang
- Department of Chemistry, Brandeis University , 415 South Street, Waltham, Massachusetts 02453, United States.,State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Collaborative Innovation Center of Chemical Science, Nankai University , Tianjin 300071, P.R. China
| | - Zhaoqianqi Feng
- Department of Chemistry, Brandeis University , 415 South Street, Waltham, Massachusetts 02453, United States
| | - Youzhi Wang
- State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Collaborative Innovation Center of Chemical Science, Nankai University , Tianjin 300071, P.R. China
| | - Rong Zhou
- Department of Chemistry, Brandeis University , 415 South Street, Waltham, Massachusetts 02453, United States
| | - Zhimou Yang
- State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Collaborative Innovation Center of Chemical Science, Nankai University , Tianjin 300071, P.R. China
| | - Bing Xu
- Department of Chemistry, Brandeis University , 415 South Street, Waltham, Massachusetts 02453, United States
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40
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Gao L, Chen J, Gao J, Wang H, Xiong W. Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes. J Cell Sci 2016; 130:396-405. [PMID: 27888215 DOI: 10.1242/jcs.192450] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 11/11/2016] [Indexed: 02/01/2023] Open
Abstract
GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F5QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F5QQI (F5QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F5QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance.
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Affiliation(s)
- Lan Gao
- State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, P.R. China.,Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R. China
| | - Junling Chen
- Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R. China.,State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilil 130022, P.R. China
| | - Jing Gao
- Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R. China.,State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilil 130022, P.R. China
| | - Hongda Wang
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilil 130022, P.R. China
| | - Wenyong Xiong
- State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, P.R. China
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41
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Jiang Y, Sverdlov MS, Toth PT, Huang LS, Du G, Liu Y, Natarajan V, Minshall RD. Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells. J Biol Chem 2016; 291:20729-38. [PMID: 27510034 DOI: 10.1074/jbc.m116.752485] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Indexed: 11/06/2022] Open
Abstract
Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.
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Affiliation(s)
- Ying Jiang
- From the School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, China, the Departments of Anesthesiology
| | | | | | - Long Shuang Huang
- Pharmacology, and Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, and
| | - Guangwei Du
- the Departments of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030
| | - Yiyao Liu
- From the School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, China
| | - Viswanathan Natarajan
- Pharmacology, and Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, and
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42
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Hamill KM, McCoy LS, Wexselblatt E, Esko JD, Tor Y. Polymyxins Facilitate Entry into Mammalian Cells. Chem Sci 2016; 7:5059-5068. [PMID: 28044098 PMCID: PMC5201209 DOI: 10.1039/c6sc00488a] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Polymyxin and guanidinylated polymyxin effectively deliver large biomolecules and liposomal assemblies into mammalian cells.
Polymyxin B is an antibiotic used against multi-resistant Gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating protein. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.
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Affiliation(s)
- Kristina M Hamill
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA
| | - Lisa S McCoy
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA
| | - Ezequiel Wexselblatt
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA
| | - Jeffrey D Esko
- Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, 92093-0687, USA
| | - Yitzhak Tor
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0358, USA
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Lorenzo DN, Healy JA, Hostettler J, Davis J, Yang J, Wang C, Hohmeier HE, Zhang M, Bennett V. Ankyrin-B metabolic syndrome combines age-dependent adiposity with pancreatic β cell insufficiency. J Clin Invest 2015; 125:3087-102. [PMID: 26168218 DOI: 10.1172/jci81317] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 05/27/2015] [Indexed: 12/22/2022] Open
Abstract
Rare functional variants of ankyrin-B have been implicated in human disease, including hereditary cardiac arrhythmia and type 2 diabetes (T2D). Here, we developed murine models to evaluate the metabolic consequences of these alterations in vivo. Specifically, we generated knockin mice that express either the human ankyrin-B variant R1788W, which is present in 0.3% of North Americans of mixed European descent and is associated with T2D, or L1622I, which is present in 7.5% of African Americans. Young AnkbR1788W/R1788W mice displayed primary pancreatic β cell insufficiency that was characterized by reduced insulin secretion in response to muscarinic agonists, combined with increased peripheral glucose uptake and concomitantly increased plasma membrane localization of glucose transporter 4 (GLUT4) in skeletal muscle and adipocytes. In contrast, older AnkbR1788W/R1788W and AnkbL1622I/L1622I mice developed increased adiposity, a phenotype that was reproduced in cultured adipocytes, and insulin resistance. GLUT4 trafficking was altered in animals expressing mutant forms of ankyrin-B, and we propose that increased cell surface expression of GLUT4 in skeletal muscle and fatty tissue of AnkbR1788W/R1788W mice leads to the observed age-dependent adiposity. Together, our data suggest that ankyrin-B deficiency results in a metabolic syndrome that combines primary pancreatic β cell insufficiency with peripheral insulin resistance and is directly relevant to the nearly one million North Americans bearing the R1788W ankyrin-B variant.
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Murfitt L, Whiteley G, Iqbal MM, Kitmitto A. Targeting caveolin-3 for the treatment of diabetic cardiomyopathy. Pharmacol Ther 2015; 151:50-71. [PMID: 25779609 DOI: 10.1016/j.pharmthera.2015.03.002] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2015] [Accepted: 03/09/2015] [Indexed: 12/21/2022]
Abstract
Diabetes is a global health problem with more than 550 million people predicted to be diabetic by 2030. A major complication of diabetes is cardiovascular disease, which accounts for over two-thirds of mortality and morbidity in diabetic patients. This increased risk has led to the definition of a diabetic cardiomyopathy phenotype characterised by early left ventricular dysfunction with normal ejection fraction. Here we review the aetiology of diabetic cardiomyopathy and explore the involvement of the protein caveolin-3 (Cav3). Cav3 forms part of a complex mechanism regulating insulin signalling and glucose uptake, processes that are impaired in diabetes. Further, Cav3 is key for stabilisation and trafficking of cardiac ion channels to the plasma membrane and so contributes to the cardiac action potential shape and duration. In addition, Cav3 has direct and indirect interactions with proteins involved in excitation-contraction coupling and so has the potential to influence cardiac contractility. Significantly, both impaired contractility and rhythm disturbances are hallmarks of diabetic cardiomyopathy. We review here how changes to Cav3 expression levels and altered relationships with interacting partners may be contributory factors to several of the pathological features identified in diabetic cardiomyopathy. Finally, the review concludes by considering ways in which levels of Cav3 may be manipulated in order to develop novel therapeutic approaches for treating diabetic cardiomyopathy.
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Affiliation(s)
- Lucy Murfitt
- Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, M13 9NT, UK
| | - Gareth Whiteley
- Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, M13 9NT, UK
| | - Mohammad M Iqbal
- Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, M13 9NT, UK
| | - Ashraf Kitmitto
- Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, M13 9NT, UK.
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Hazan-Halevy I, Rosenblum D, Weinstein S, Bairey O, Raanani P, Peer D. Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes. Cancer Lett 2015; 364:59-69. [PMID: 25933830 DOI: 10.1016/j.canlet.2015.04.026] [Citation(s) in RCA: 119] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2015] [Revised: 04/22/2015] [Accepted: 04/23/2015] [Indexed: 12/14/2022]
Abstract
Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies.
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Affiliation(s)
- Inbal Hazan-Halevy
- Laboratory of NanoMedicine, Department of Cell Research & Immunology, Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel
| | - Daniel Rosenblum
- Laboratory of NanoMedicine, Department of Cell Research & Immunology, Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel
| | - Shiri Weinstein
- Laboratory of NanoMedicine, Department of Cell Research & Immunology, Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel
| | - Osnat Bairey
- Institute of Hematology, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel
| | - Pia Raanani
- Institute of Hematology, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel
| | - Dan Peer
- Laboratory of NanoMedicine, Department of Cell Research & Immunology, Department of Materials Science and Engineering, Tel Aviv University, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel.
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Ji S, Ohkawa Y, Tokizane K, Ohmi Y, Banno R, Furukawa K, Kiyama H, Furukawa K. b-series gangliosides crucially regulate leptin secretion in adipose tissues. Biochem Biophys Res Commun 2015; 459:189-195. [DOI: 10.1016/j.bbrc.2015.01.143] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Accepted: 01/29/2015] [Indexed: 12/11/2022]
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Llanos P, Contreras-Ferrat A, Georgiev T, Osorio-Fuentealba C, Espinosa A, Hidalgo J, Hidalgo C, Jaimovich E. The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice. Am J Physiol Endocrinol Metab 2015; 308:E294-305. [PMID: 25491723 DOI: 10.1152/ajpendo.00189.2014] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MβCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MβCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MβCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MβCD on glucose uptake. Addition of MβCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.
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Affiliation(s)
- Paola Llanos
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile;
| | - Ariel Contreras-Ferrat
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Institute for Research in Dental Sciences, Facultad de Odontología, Universidad de Chile, Santiago, Chile
| | - Tihomir Georgiev
- Medical Biophysics, Institute of Physiology und Pathophysiology, Ruprecht Karls Universität, Heidelberg, Germany
| | | | - Alejandra Espinosa
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile
| | - Jorge Hidalgo
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Physiology and Biophysics Program, Institute of Biomedical Sciences (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile
| | - Cecilia Hidalgo
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Physiology and Biophysics Program, Institute of Biomedical Sciences (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; Biomedical Neuroscience Institute, Facultad de Medicina, Universidad de Chile, Santiago, Chile; and
| | - Enrique Jaimovich
- Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Cell and Molecular Biology Program, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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Govers R. Molecular mechanisms of GLUT4 regulation in adipocytes. DIABETES & METABOLISM 2014; 40:400-10. [PMID: 24656589 DOI: 10.1016/j.diabet.2014.01.005] [Citation(s) in RCA: 92] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Revised: 01/24/2014] [Accepted: 01/26/2014] [Indexed: 01/28/2023]
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Watanabe S, Hayakawa T, Wakasugi K, Yamanaka K. Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity. Cell Death Dis 2014; 5:e1497. [PMID: 25356866 PMCID: PMC4237269 DOI: 10.1038/cddis.2014.459] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2014] [Revised: 07/23/2014] [Accepted: 08/25/2014] [Indexed: 12/13/2022]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies observed in the spinal motor neurons of sporadic ALS and is decreased in the cerebrospinal fluid of ALS patients. Despite prominent deposition of CysC in ALS, the roles of CysC in the central nervous system remain unknown. Here, we identified the neuroprotective activity of CysC against ALS-linked mutant Cu/Zn-superoxide dismutase (SOD1)-mediated toxicity. We found that exogenously added CysC protected neuronal cells including primary cultured motor neurons. Moreover, the neuroprotective property of CysC was dependent on the coordinated activation of two distinct pathways: autophagy induction through AMPK-mTOR pathway and inhibition of cathepsin B. Furthermore, exogenously added CysC was transduced into the cells and aggregated in the cytosol under oxidative stress conditions, implying a relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest CysC is an endogenous neuroprotective agent and targeting CysC in motor neurons may provide a novel therapeutic strategy for ALS.
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Affiliation(s)
- S Watanabe
- 1] Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, Japan [2] Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan
| | - T Hayakawa
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan
| | - K Wakasugi
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan
| | - K Yamanaka
- 1] Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, Japan [2] Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan
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