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Park JH, Jeong EY, Kim YH, Cha SY, Kim HY, Nam YK, Park JS, Kim SY, Lee YJ, Yoon JH, So B, Kim D, Kim M, Byun Y, Lee YH, Shin SS, Park JT. Epigallocatechin Gallate in Camellia sinensis Ameliorates Skin Aging by Reducing Mitochondrial ROS Production. Pharmaceuticals (Basel) 2025; 18:612. [PMID: 40430436 PMCID: PMC12114381 DOI: 10.3390/ph18050612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2025] [Revised: 04/18/2025] [Accepted: 04/21/2025] [Indexed: 05/29/2025] Open
Abstract
Background: Reactive oxygen species (ROS) generated by mitochondrial dysfunction damage cellular organelles and contribute to skin aging. Therefore, strategies to reduce mitochondrial ROS production are considered important for alleviating skin aging, but no effective methods have been identified. Methods: In this study, we evaluated substances utilized as cosmetic ingredients and discovered Camellia sinensis (C. sinensis) as a substance that reduces mitochondrial ROS levels. Results:C. sinensis extracts were found to act as senolytics that selectively kill senescent fibroblasts containing dysfunctional mitochondria. In addition, C. sinensis extracts facilitated efficient electron transport in the mitochondrial electron transport chain (ETC) by increasing the efficiency of oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ROS production, a byproduct of the inefficient ETC. This novel mechanism of C. sinensis extracts led to the restoration of skin aging and the skin barrier. Furthermore, epigallocatechin gallate (EGCG) was identified as an active ingredient that plays a key role in C. sinensis extract-mediated skin aging recovery. Indeed, similar to C. sinensis extracts, EGCG reduced ROS and improved skin aging in an artificial skin model. Conclusions: Our data uncovered a novel mechanism by which C. sinensis extract reverses skin aging by reducing mitochondrial ROS production via selective senescent cell death/increased OXPHOS efficiency. Our results suggest that C. sinensis extract or EGCG may be used as a therapeutic agent to reverse skin aging in clinical and cosmetic applications.
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Affiliation(s)
- Ji Ho Park
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Eun Young Jeong
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Ye Hyang Kim
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - So Yoon Cha
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Ha Yeon Kim
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Yeon Kyung Nam
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Jin Seong Park
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - So Yeon Kim
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Yoo Jin Lee
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Jee Hee Yoon
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Byeonghyeon So
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Duyeol Kim
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Minseon Kim
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Youngjoo Byun
- College of Pharmacy, Korea University, Sejong 30019, Republic of Korea;
- Interdisciplinary Major Program in Innovative Pharmaceutical Sciences, Korea University, Sejong 30019, Republic of Korea
| | - Yun Haeng Lee
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
| | - Song Seok Shin
- Hyundai Bioland Co., Ltd., 22, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si 28162, Republic of Korea; (E.Y.J.); (Y.H.K.); (S.Y.C.); (H.Y.K.); (Y.K.N.); (J.S.P.); (S.Y.K.)
| | - Joon Tae Park
- Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea; (J.H.P.); (Y.J.L.); (J.H.Y.); (B.S.); (D.K.); (M.K.)
- Convergence Research Center for Insect Vectors, Incheon National University, Incheon 22012, Republic of Korea
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Schmidt L, Saynisch M, Hoegsbjerg C, Schmidt A, Mackey A, Lackmann JW, Müller S, Koch M, Brachvogel B, Kjaer M, Antczak P, Krüger M. Spatial proteomics of skeletal muscle using thin cryosections reveals metabolic adaptation at the muscle-tendon transition zone. Cell Rep 2024; 43:114374. [PMID: 38900641 DOI: 10.1016/j.celrep.2024.114374] [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: 12/07/2023] [Revised: 04/05/2024] [Accepted: 05/31/2024] [Indexed: 06/22/2024] Open
Abstract
Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.
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Affiliation(s)
- Luisa Schmidt
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
| | - Michael Saynisch
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
| | - Christian Hoegsbjerg
- Institute of Sports Medicine Copenhagen, Department of Orthopaedic Surgery, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark; Part of IOC Research Center Copenhagen and Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | | | - Abigail Mackey
- Institute of Sports Medicine Copenhagen, Department of Orthopaedic Surgery, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark; Part of IOC Research Center Copenhagen and Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jan-Wilm Lackmann
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
| | - Stefan Müller
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
| | - Manuel Koch
- Institute for Dental Research and Oral Musculoskeletal Biology, Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany
| | - Bent Brachvogel
- Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany; Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany
| | - Michael Kjaer
- Institute of Sports Medicine Copenhagen, Department of Orthopaedic Surgery, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark; Part of IOC Research Center Copenhagen and Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Philipp Antczak
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany.
| | - Marcus Krüger
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany.
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3
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Fan Y, Pionneau C, Cocozza F, Boëlle P, Chardonnet S, Charrin S, Théry C, Zimmermann P, Rubinstein E. Differential proteomics argues against a general role for CD9, CD81 or CD63 in the sorting of proteins into extracellular vesicles. J Extracell Vesicles 2023; 12:e12352. [PMID: 37525398 PMCID: PMC10390663 DOI: 10.1002/jev2.12352] [Citation(s) in RCA: 45] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 07/15/2023] [Indexed: 08/02/2023] Open
Abstract
The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.
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Affiliation(s)
- Yé Fan
- Centre d'Immunologie et des Maladies InfectieusesSorbonne Université, Inserm, CNRSParisFrance
| | - Cédric Pionneau
- UMS Production et Analyse des données en Sciences de la vie et en Santé, PASSPlateforme Post‐génomique de la Pitié‐Salpêtrière, P3SSorbonne Université, InsermParisFrance
| | - Federico Cocozza
- Inserm U932, Institut Curie Centre de RecherchePSL Research UniversityParisFrance
| | - Pierre‐Yves Boëlle
- Institut Pierre Louis d’Épidémiologie et de Santé PubliqueSorbonne Université, InsermParisFrance
| | - Solenne Chardonnet
- UMS Production et Analyse des données en Sciences de la vie et en Santé, PASSPlateforme Post‐génomique de la Pitié‐Salpêtrière, P3SSorbonne Université, InsermParisFrance
| | - Stéphanie Charrin
- Centre d'Immunologie et des Maladies InfectieusesSorbonne Université, Inserm, CNRSParisFrance
| | - Clotilde Théry
- Inserm U932, Institut Curie Centre de RecherchePSL Research UniversityParisFrance
- CurieCoretech Extracellular VesiclesInstitut Curie Centre de RechercheParisFrance
| | - Pascale Zimmermann
- Centre de Recherche en Cancérologie de Marseille (CRCM)Institut Paoli‐Calmettes, Aix‐Marseille Université, Inserm, CNRSMarseilleFrance
- Department of Human GeneticsKatholieke Universiteit Leuven (KU Leuven)LeuvenBelgium
| | - Eric Rubinstein
- Centre d'Immunologie et des Maladies InfectieusesSorbonne Université, Inserm, CNRSParisFrance
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Ding Y, Chen J, Li S, Wren JD, Bajpai AK, Wang J, Tanaka T, Rice HC, Hays FA, Lu L, Zhang XA. EWI2 and its relatives in Tetraspanin-enriched membrane domains regulate malignancy. Oncogene 2023; 42:861-868. [PMID: 36788350 DOI: 10.1038/s41388-023-02623-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Revised: 01/24/2023] [Accepted: 02/01/2023] [Indexed: 02/16/2023]
Abstract
Experimental studies on immunoglobulin superfamily (IgSF) member EWI2 reveal that it suppresses a variety of solid malignant tumors including brain, lung, skin, and prostate cancers in animal models and inhibits tumor cell movement and growth in vitro. While EWI2 appears to support myeloid leukemia in mouse models and maintain leukemia stem cells. Bioinformatics analyses suggest that EWI2 gene expression is downregulated in glioblastoma but upregulated in melanoma, pancreatic cancer, and liver cancer. The mechanism of action for EWI2 is linked to its inhibition of growth factor receptors and cell adhesion proteins through its associated tetraspanin-enriched membrane domains (TEMDs), by altering the cell surface clustering and endolysosome trafficking/turnover of these transmembrane proteins. Recent studies also show that EWI2 modulates the nuclear translocation of ERK and TFEB to change the activities of these gene expression regulators. For EWI2 relatives including FPRP, IgSF3, and CD101, although their roles in malignant diseases are not fully clear and remain to be determined experimentally, FPRP and IgSF3 likely promote the progression of solid malignant tumors while CD101 seems to modulate immune cells of tumor microenvironment. Distinctive from other tumor regulators, the impacts of EWI subfamily members on solid malignant tumors are likely to be context dependent. In other words, the effect of a given EWI subfamily member on a tumor probably depends on the molecular network and composition of TEMDs in that tumor. Collectively, EWI2 and its relatives are emerged as important regulators of malignant diseases with promising potentials to become anti-cancer therapeutics and cancer therapy targets.
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Affiliation(s)
- Yingjun Ding
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Junxiong Chen
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Shuping Li
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Jonathan D Wren
- Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
| | | | - Jie Wang
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Takemi Tanaka
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Heather C Rice
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Franklin A Hays
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
| | - Lu Lu
- University of Tennessee Health Science Center, Memphis, TN, USA
| | - Xin A Zhang
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
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5
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Abstract
Single-pass transmembrane receptors (SPTMRs) represent a diverse group of integral membrane proteins that are involved in many essential cellular processes, including signal transduction, cell adhesion, and transmembrane transport of materials. Dysregulation of the SPTMRs is linked with many human diseases. Despite extensive efforts in past decades, the mechanisms of action of the SPTMRs remain incompletely understood. One major hurdle is the lack of structures of the full-length SPTMRs in different functional states. Such structural information is difficult to obtain by traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR). The recent rapid development of single-particle cryo-electron microscopy (cryo-EM) has led to an exponential surge in the number of high-resolution structures of integral membrane proteins, including SPTMRs. Cryo-EM structures of SPTMRs solved in the past few years have tremendously improved our understanding of how SPTMRs function. In this review, we will highlight these progresses in the structural studies of SPTMRs by single-particle cryo-EM, analyze important structural details of each protein involved, and discuss their implications on the underlying mechanisms. Finally, we also briefly discuss remaining challenges and exciting opportunities in the field.
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Affiliation(s)
- Kai Cai
- Departments of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas 75231, USA
| | - Xuewu Zhang
- Departments of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas 75231, USA
- Departments of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75231, USA
- Corresponding Author: Xuewu Zhang, Department of pharmacology, UT Southwestern Medical Center, Dallas, TX 75390, USA;
| | - Xiao-chen Bai
- Departments of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas 75231, USA
- Departments of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75231, USA
- Corresponding Author: Xiao-chen Bai, Department of Biophysics, UT Southwestern Medical Center, Dallas, TX 75390, USA;
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Wang J, Wren JD, Ding Y, Chen J, Mittal N, Xu C, Li X, Zeng C, Wang M, Shi J, Zhang YH, Han SJ, Zhang XA. EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer. Cancer Lett 2022; 536:215641. [PMID: 35339615 PMCID: PMC9036562 DOI: 10.1016/j.canlet.2022.215641] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 03/07/2022] [Accepted: 03/10/2022] [Indexed: 11/30/2022]
Abstract
As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.
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Affiliation(s)
- Jie Wang
- University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Jonathan D. Wren
- Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
| | - Yingjun Ding
- University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Junxiong Chen
- University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Nikhil Mittal
- Michigan Technological University, Houghton, Michigan, USA
| | - Chao Xu
- University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Xing Li
- Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Cengxi Zeng
- Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Meng Wang
- Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Jing Shi
- Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Yanhui H. Zhang
- University of Tennessee Health Sciences Center, Memphis, Tennessee, USA
| | | | - Xin A. Zhang
- University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA,To whom correspondence should be addressed: Dr. Xin Zhang, Stephenson Cancer Center and Department of Physiology, University of Oklahoma Health Sciences Center, Biomedical Research Center Room 1474, 975 NE 10 Street, Oklahoma City, OK 73104. Tel: 405-271-8001 (ext. 56218);
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7
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Jimbo K, Nakajima-Takagi Y, Ito T, Koide S, Nannya Y, Iwama A, Tojo A, Konuma T. Immunoglobulin superfamily member 8 maintains myeloid leukemia stem cells through inhibition of β-catenin degradation. Leukemia 2022; 36:1550-1562. [PMID: 35418614 DOI: 10.1038/s41375-022-01564-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 03/09/2022] [Accepted: 03/30/2022] [Indexed: 11/10/2022]
Abstract
The identification of characteristic differences between cancer stem cells and their normal counterparts remains a key challenge for cancer treatment. Here, we investigated the role of immunoglobulin superfamily member 8 (Igsf8, also known as EWI-2, PGRL, and CD316) on normal and malignant hematopoietic stem cells, mainly using the conditional knockout model. Deletion of Igsf8 did not affect steady state hematopoiesis, but it led to a significant improvement of survival in mouse myeloid leukemia models. Deletion of Igsf8 significantly depletes leukemia stem cells (LSCs) through enhanced apoptosis and β-catenin degradation. At a molecular level, we found that activation of β-catenin in LSCs depends on Igsf8, which promotes the association of FZD4 with its co-receptor LRP6 in the presence of Igsf8. Similarly, IGSF8 inhibition blocks the colony-forming ability of LSCs and improves the survival of recipients in xenograft models of myeloid leukemia. Collectively, these data indicate strong genetic evidence identifying Igsf8 as a key regulator of myeloid leukemia and the possibility that targeting IGSF8 may serve as a new therapeutic approach against myeloid leukemia.
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Affiliation(s)
- Koji Jimbo
- Division of Hematopoietic Disease Control, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,Division of Stem Cell and Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Yaeko Nakajima-Takagi
- Division of Stem Cell and Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Takahiro Ito
- Laboratory of Cell Fate Dynamics and Therapeutics, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Shuhei Koide
- Division of Stem Cell and Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Yasuhito Nannya
- Division of Hematopoietic Disease Control, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Atsushi Iwama
- Division of Stem Cell and Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Arinobu Tojo
- Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Takaaki Konuma
- Division of Hematopoietic Disease Control, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. .,Department of Hematology/Oncology, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
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8
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P C S, Shetty SS, Nalilu SK, Shetty PK, Patil P. Tetraspanin CD9: A friend or foe of head and neck cancer (Review). Oncol Rep 2022; 47:88. [PMID: 35266009 PMCID: PMC8931833 DOI: 10.3892/or.2022.8299] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Accepted: 11/15/2021] [Indexed: 12/02/2022] Open
Abstract
Head and neck cancers are diverse and complex diseases characterised by unregulated growth of tumour cells in various parts of the head and neck region, such as in the buccal mucosa, floor of the mouth, tongue, oropharynx, hypopharynx, oesophagus, nasopharynx and salivary glands. Partial or total glossectomy, radiation or chemotherapy greatly affect patient quality of life. However, even following treatment, patients may relapse. Nicotine-derived nitrosamines and alcohol are the major etiological factors underlying this deadly disease. These compounds induce DNA damage that may lead to mutation in crucial genes, such as p53 and p21, which are important to regulate cell proliferation, thus leading to cancer. CD9 is a tetraspanin, which are a group of transmembrane proteins that have a role in cell motility and adhesion. The present review aimed to explore the role of CD9 in head and neck cancer. Epidermal growth factor receptor activity and cell proliferation are regulated by the CD9-integrin/CD9-transforming growth factor interaction. Hence, CD9 can play a dual role in various types of cancer.
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Affiliation(s)
- Suhasini P C
- Central Research Laboratory, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka 575018, India
| | - Shilpa S Shetty
- Central Research Laboratory, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka 575018, India
| | - Suchetha Kumari Nalilu
- Department of Biochemistry, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka 575018, India
| | - Praveen Kumar Shetty
- Department of Biochemistry, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka 575018, India
| | - Prakash Patil
- Central Research Laboratory, K.S. Hegde Medical Academy, Nitte (Deemed to be University), Mangalore, Karnataka 575018, India
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Sun G, Chen J, Ding Y, Wren JD, Xu F, Lu L, Wang Y, Wang DW, Zhang XA. A Bioinformatics Perspective on the Links Between Tetraspanin-Enriched Microdomains and Cardiovascular Pathophysiology. Front Cardiovasc Med 2021; 8:630471. [PMID: 33860000 PMCID: PMC8042132 DOI: 10.3389/fcvm.2021.630471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 02/15/2021] [Indexed: 11/13/2022] Open
Abstract
Background: Tetraspanins and integrins are integral membrane proteins. Tetraspanins interact with integrins to modulate the dynamics of adhesion, migration, proliferation, and signaling in the form of membrane domains called tetraspanin-enriched microdomains (TEMs). TEMs also contain other cell adhesion proteins like immunoglobulin superfamily (IgSF) proteins and claudins. Cardiovascular functions of these TEM proteins have emerged and remain to be further revealed. Objectives: The aims of this study are to explore the roles of these TEM proteins in the cardiovascular system using bioinformatics tools and databases and to highlight the TEM proteins that may functionally associate with cardiovascular physiology and pathology. Methods: For human samples, three databases-GTEx, NCBI-dbGaP, and NCBI-GEO-were used for the analyses. The dbGaP database was used for GWAS analysis to determine the association between target genes and human phenotypes. GEO is an NCBI public repository that archives genomics data. GTEx was used for the analyses of tissue-specific mRNA expression levels and eQTL. For murine samples, GeneNetwork was used to find gene-phenotype correlations and gene-gene correlations of expression levels in mice. The analysis of cardiovascular data was the focus of this study. Results: Some integrins and tetraspanins, such as ITGA8 and Cd151, are highly expressed in the human cardiovascular system. TEM components are associated with multiple cardiovascular pathophysiological events in humans. GWAS and GEO analyses showed that human Cd82 and ITGA9 are associated with blood pressure. Data from mice also suggest that various cardiovascular phenotypes are correlated with integrins and tetraspanins. For instance, Cd82 and ITGA9, again, have correlations with blood pressure in mice. Conclusion: ITGA9 is related to blood pressure in both species. KEGG analysis also linked ITGA9 to metabolism and MAPK signaling pathway. This work provides an example of using integrated bioinformatics approaches across different species to identify the connections of structurally and/or functionally related molecules to certain categories of diseases.
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Affiliation(s)
- Ge Sun
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Junxiong Chen
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Yingjun Ding
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Jonathan D. Wren
- Oklahoma Medical Research Foundation, Oklahoma City, OK, United States
| | - Fuyi Xu
- University of Tennessee Health Science Center, Memphis, TN, United States
| | - Lu Lu
- University of Tennessee Health Science Center, Memphis, TN, United States
| | - Yan Wang
- Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China
| | - Dao-wen Wang
- Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China
| | - Xin A. Zhang
- University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
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Fu C, Zhang Q, Wang A, Yang S, Jiang Y, Bai L, Wei Q. EWI-2 controls nucleocytoplasmic shuttling of EGFR signaling molecules and miRNA sorting in exosomes to inhibit prostate cancer cell metastasis. Mol Oncol 2021; 15:1543-1565. [PMID: 33605506 PMCID: PMC8096798 DOI: 10.1002/1878-0261.12930] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 01/18/2021] [Accepted: 02/17/2021] [Indexed: 02/05/2023] Open
Abstract
Early and accurate diagnosis of prostate cancer (PCa) is extremely important, as metastatic PCa remains hard to treat. EWI-2, a member of the Ig protein subfamily, is known to inhibit PCa cell migration. In this study, we found that EWI-2 localized on both the cell membrane and exosomes regulates the distribution of miR-3934-5p between cells and exosomes. Interestingly, we observed that EWI-2 is localized not only on the plasma membrane but also on the nuclear envelope (nuclear membrane), where it regulates the nuclear translocation of signaling molecules and miRNA. Collectively, these functions of EWI-2 found in lipid bilayers appear to regulate PCa cell metastasis through the epidermal growth factor receptor-mitogen-activated protein kinase-extracellular-signal-regulated kinase (EGFR-MAPK-ERK) pathway. Our research provides new insights into the molecular function of EWI-2 on PCa metastasis, and highlights EWI-2 as a potential PCa biomarker.
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Affiliation(s)
- Chenying Fu
- State Key Laboratory of Biotherapy and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Qing Zhang
- Department of Rehabilitation Medicine Center, Key Laboratory of Rehabilitation Medicine in Sichuan Province, West China Hospital, Sichuan University, Chengdu, China
| | - Ani Wang
- Cadiovascular Center, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China
| | - Songpeng Yang
- State Key Laboratory of Biotherapy and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yangfu Jiang
- State Key Laboratory of Biotherapy and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Lin Bai
- Research Core Facility, West China Hospital, Sichuan University, Chengdu, China
| | - Quan Wei
- State Key Laboratory of Biotherapy and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China.,Department of Rehabilitation Medicine Center, Key Laboratory of Rehabilitation Medicine in Sichuan Province, West China Hospital, Sichuan University, Chengdu, China
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11
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Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer. Nat Commun 2020; 11:5171. [PMID: 33057002 PMCID: PMC7560607 DOI: 10.1038/s41467-020-18956-x] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Accepted: 09/23/2020] [Indexed: 12/20/2022] Open
Abstract
Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function. Mossy fiber synapses are key in CA3 microcircuit function. Here, the authors profile the mossy fiber synapse proteome and cell-surface interactome. They uncover a diverse repertoire of cell-surface proteins and identify the receptor IgSF8 as a regulator of CA3 microcircuit connectivity and function.
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12
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Kummer D, Steinbacher T, Schwietzer MF, Thölmann S, Ebnet K. Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease. Med Microbiol Immunol 2020; 209:397-405. [PMID: 32274581 PMCID: PMC7395057 DOI: 10.1007/s00430-020-00673-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 03/28/2020] [Indexed: 12/27/2022]
Abstract
Tetraspanins comprise a family of proteins embedded in the membrane through four transmembrane domains. One of the most distinctive features of tetraspanins is their ability to interact with other proteins in the membrane using their extracellular, transmembrane and cytoplasmic domains, allowing them to incorporate several proteins into clusters called tetraspanin-enriched microdomains. The spatial proximity of signaling proteins and their regulators enables a rapid functional cross-talk between these proteins, which is required for a rapid translation of extracellular signals into intracellular signaling cascades. In this article, we highlight a few examples that illustrate how tetraspanin-mediated interactions between cell surface proteins allow their functional cross-talk to regulate intracellular signaling.
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Affiliation(s)
- Daniel Kummer
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Münster, Germany
- Interdisciplinary Clinical Research Center (IZKF), University of Münster, Münster, Germany
| | - Tim Steinbacher
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Münster, Germany
- Cells-In-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, Münster, Germany
| | - Mariel Flavia Schwietzer
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Münster, Germany
| | - Sonja Thölmann
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Münster, Germany
| | - Klaus Ebnet
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Münster, Germany.
- Interdisciplinary Clinical Research Center (IZKF), University of Münster, Münster, Germany.
- Cells-In-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, Münster, Germany.
- Institute of Medical Biochemistry, ZMBE, Von-Esmarch-Str. 56, 48149, Münster, Germany.
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Jankovičová J, Neuerová Z, Sečová P, Bartóková M, Bubeníčková F, Komrsková K, Postlerová P, Antalíková J. Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos. Med Microbiol Immunol 2020; 209:407-425. [PMID: 32424440 DOI: 10.1007/s00430-020-00676-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Accepted: 05/02/2020] [Indexed: 12/21/2022]
Abstract
It is known that tetraspanin proteins are involved in many physiological somatic cell mechanisms. Additionally, research has indicated they also have a role in various infectious diseases and cancers. This review focuses on the molecular interactions underlying the tetraspanin web formation in gametes. Primarily, tetraspanins act in the reproductive tract as organizers of membrane complexes, which include the proteins involved in the contact and association of sperm and oocyte membranes. In addition, recent data shows that tetraspanins are likely to be involved in these processes in a complex way. In mammalian fertilization, an important role is attributed to CD molecules belonging to the tetraspanin superfamily, particularly CD9, CD81, CD151, and also CD63; mostly as part of extracellular vesicles, the significance of which and their potential in reproduction is being intensively investigated. In this article, we reviewed the existing knowledge regarding the expression of tetraspanins CD9, CD81, CD151, and CD63 in mammalian spermatozoa, oocytes, and embryos and their involvement in reproductive processes, including pathological events.
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Affiliation(s)
- Jana Jankovičová
- Laboratory of Reproductive Physiology, Center of Biosciences, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Zdeňka Neuerová
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic
- Faculty of Science, University of Hradec Králové, Hradec Králové, Czech Republic
| | - Petra Sečová
- Laboratory of Reproductive Physiology, Center of Biosciences, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Michaela Bartóková
- Laboratory of Reproductive Physiology, Center of Biosciences, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Filipa Bubeníčková
- Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic
| | - Kateřina Komrsková
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic
- Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Pavla Postlerová
- Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic
- Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic
| | - Jana Antalíková
- Laboratory of Reproductive Physiology, Center of Biosciences, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.
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Umeda R, Satouh Y, Takemoto M, Nakada-Nakura Y, Liu K, Yokoyama T, Shirouzu M, Iwata S, Nomura N, Sato K, Ikawa M, Nishizawa T, Nureki O. Structural insights into tetraspanin CD9 function. Nat Commun 2020; 11:1606. [PMID: 32231207 PMCID: PMC7105497 DOI: 10.1038/s41467-020-15459-7] [Citation(s) in RCA: 130] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2019] [Accepted: 03/06/2020] [Indexed: 01/16/2023] Open
Abstract
Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
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Affiliation(s)
- Rie Umeda
- Department of Biological Sciences Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
| | - Yuhkoh Satouh
- Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi, 371-8512, Japan
- Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan
| | - Mizuki Takemoto
- Department of Biological Sciences Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
- Preferred Networks, Inc., Bunkyo-ku, Tokyo, Japan
| | - Yoshiko Nakada-Nakura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kehong Liu
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takeshi Yokoyama
- Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
| | - Mikako Shirouzu
- Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
| | - So Iwata
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo, Japan
| | - Norimichi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ken Sato
- Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi, 371-8512, Japan
- Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi, 371-8512, Japan
| | - Masahito Ikawa
- Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan
| | - Tomohiro Nishizawa
- Department of Biological Sciences Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
- Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology, Bunkyo-ku, Tokyo, Japan.
| | - Osamu Nureki
- Department of Biological Sciences Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
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15
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Zheng T, Yang J. Differential expression of EWI-2 in endometriosis, its functional role and underlying molecular mechanisms. J Obstet Gynaecol Res 2017; 43:1180-1188. [PMID: 28544021 DOI: 10.1111/jog.13333] [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] [Received: 11/11/2016] [Revised: 02/09/2017] [Accepted: 02/26/2017] [Indexed: 12/25/2022]
Abstract
AIM We aimed to investigate EWI-2 expression in endometrium tissues collected from women with endometriosis at mRNA and protein levels, to evaluate its potential as a biomarker for endometriosis and to study its functional role via possible regulation of the PI3K/Akt signaling pathway. METHODS Endometrium tissues were collected from patients with endometriosis and healthy individuals. EWI-2 mRNA expression was evaluated using quantitative real-time PCR (qRT-PCR) while EWI-2 protein levels were determined by western blotting. For functional studies, EWI-2 shRNA was transfected in endometrial epithelial cells and the in vitro migration and invasion assays were performed using the Transwell chambers. RESULTS EWI-2 was significantly downregulated in tissues obtained from patients with endometriosis compared with healthy individuals (P < 0.0001). EWI-2 expression in the secretory phase was lower than that in the proliferative phase (P < 0.0001). Receiver-operator curve analysis of EWI-2 expression showed that the area under the curve for endometriosis diagnosis was 0.8942 (P = 0.003), 0.9643 (P = 0.0001), 0.9912 (P < 0.0001), and 0.9150 (P < 0.0001), respectively, for healthy women compared with women with endometriosis in matched comparisons of data originated from the proliferative, early, middle, and late secretory phases. Over the menstrual cycle, the expression of EWI-2 was significantly decreased in the eutopic tissues compared to the ectopic tissues. Further cellular and molecular analyses showed that EWI-2 inhibited cell migration and invasion via the Akt signaling. CONCLUSION Our findings suggested that downregulation of EWI-2 may contribute to endometriosis physiopathology and potentiate EWI-2 as a valuable diagnostic biomarker and therapeutic target for endometriosis.
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Affiliation(s)
- Tingting Zheng
- Reproductive Medicine Center, Renmin Hospital of Wuhan University, Wuhan City, Hubei, China
| | - Jing Yang
- Reproductive Medicine Center, Renmin Hospital of Wuhan University, Wuhan City, Hubei, China
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16
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Usardi A, Iyer K, Sigoillot SM, Dusonchet A, Selimi F. The immunoglobulin-like superfamily member IGSF3 is a developmentally regulated protein that controls neuronal morphogenesis. Dev Neurobiol 2016; 77:75-92. [PMID: 27328461 DOI: 10.1002/dneu.22412] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 06/18/2016] [Accepted: 06/18/2016] [Indexed: 01/06/2023]
Abstract
The establishment of a functional brain depends on the fine regulation and coordination of many processes, including neurogenesis, differentiation, dendritogenesis, axonogenesis, and synaptogenesis. Proteins of the immunoglobulin-like superfamily (IGSF) are major regulators during this sequence of events. Different members of this class of proteins play nonoverlapping functions at specific developmental time-points, as shown in particular by studies of the cerebellum. We have identified a member of the little studied EWI subfamily of IGSF, the protein IGSF3, as a membrane protein expressed in a neuron specific- and time-dependent manner during brain development. In the cerebellum, it is transiently found in membranes of differentiating granule cells, and is particularly concentrated at axon terminals. There it co-localizes with other IGSF proteins with well-known functions in cerebellar development: TAG-1 and L1. Functional analysis shows that IGSF3 controls the differentiation of granule cells, more precisely axonal growth and branching. Biochemical experiments demonstrate that, in the developing brain, IGSF3 is in a complex with the tetraspanin TSPAN7, a membrane protein mutated in several forms of X-linked intellectual disabilities. In cerebellar granule cells, TSPAN7 promotes axonal branching and the size of TSPAN7 clusters is increased by downregulation of IGSF3. Thus IGSF3 is a novel regulator of neuronal morphogenesis that might function through interactions with multiple partners including the tetraspanin TSPAN7. This developmentally regulated protein might thus be at the center of a new signaling pathway controlling brain development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 75-92, 2017.
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Affiliation(s)
- Alessia Usardi
- Team Mice, Molecules and Synapse Formation, CIRB, Collège de France, CNRS, INSERM, PSL* Research University, Paris, France, 75231, Cedex 05
| | - Keerthana Iyer
- Team Mice, Molecules and Synapse Formation, CIRB, Collège de France, CNRS, INSERM, PSL* Research University, Paris, France, 75231, Cedex 05
| | - Séverine M Sigoillot
- Team Mice, Molecules and Synapse Formation, CIRB, Collège de France, CNRS, INSERM, PSL* Research University, Paris, France, 75231, Cedex 05
| | - Antoine Dusonchet
- Team Mice, Molecules and Synapse Formation, CIRB, Collège de France, CNRS, INSERM, PSL* Research University, Paris, France, 75231, Cedex 05
| | - Fekrije Selimi
- Team Mice, Molecules and Synapse Formation, CIRB, Collège de France, CNRS, INSERM, PSL* Research University, Paris, France, 75231, Cedex 05
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Detchokul S, Williams ED, Parker MW, Frauman AG. Tetraspanins as regulators of the tumour microenvironment: implications for metastasis and therapeutic strategies. Br J Pharmacol 2015; 171:5462-90. [PMID: 23731188 DOI: 10.1111/bph.12260] [Citation(s) in RCA: 72] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2013] [Revised: 05/16/2013] [Accepted: 05/16/2013] [Indexed: 12/13/2022] Open
Abstract
UNLABELLED One of the hallmarks of cancer is the ability to activate invasion and metastasis. Cancer morbidity and mortality are largely related to the spread of the primary, localized tumour to adjacent and distant sites. Appropriate management and treatment decisions based on predicting metastatic disease at the time of diagnosis is thus crucial, which supports better understanding of the metastatic process. There are components of metastasis that are common to all primary tumours: dissociation from the primary tumour mass, reorganization/remodelling of extracellular matrix, cell migration, recognition and movement through endothelial cells and the vascular circulation and lodgement and proliferation within ectopic stroma. One of the key and initial events is the increased ability of cancer cells to move, escaping the regulation of normal physiological control. The cellular cytoskeleton plays an important role in cancer cell motility and active cytoskeletal rearrangement can result in metastatic disease. This active change in cytoskeletal dynamics results in manipulation of plasma membrane and cellular balance between cellular adhesion and motility which in turn determines cancer cell movement. Members of the tetraspanin family of proteins play important roles in regulation of cancer cell migration and cancer-endothelial cell interactions, which are critical for cancer invasion and metastasis. Their involvements in active cytoskeletal dynamics, cancer metastasis and potential clinical application will be discussed in this review. In particular, the tetraspanin member, CD151, is highlighted for its major role in cancer invasion and metastasis. LINKED ARTICLES This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24.
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Affiliation(s)
- S Detchokul
- Clinical Pharmacology and Therapeutics Unit, Department of Medicine (Austin Health/Northern Health), The University of Melbourne, Heidelberg, Vic., Australia
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Murayama Y, Oritani K, Tsutsui S. Novel CD9-targeted therapies in gastric cancer. World J Gastroenterol 2015; 21:3206-3213. [PMID: 25805926 PMCID: PMC4363749 DOI: 10.3748/wjg.v21.i11.3206] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2014] [Revised: 11/13/2014] [Accepted: 12/16/2014] [Indexed: 02/06/2023] Open
Abstract
There are 33 human tetraspanin proteins, emerging as key players in malignancy, the immune system, fertilization, cellular signaling, adhesion, morphology, motility, proliferation, and tumor invasion. CD9, a member of the tetraspanin family, associates with and influences a variety of cell-surface molecules. Through these interactions, CD9 modifies multiple cellular events, including adhesion, migration, proliferation, and survival. CD9 is therefore considered to play a role in several stages during cancer development. Reduced CD9 expression is generally related to venous vessel invasion and metastasis as well as poor prognosis. We found that treatment of mice bearing human gastric cancer cells with anti-CD9 antibody successfully inhibited tumor progression via antiproliferative, proapoptotic, and antiangiogenic effects, strongly indicating that CD9 is a possible therapeutic target in patients with gastric cancer. Here, we describe the possibility of CD9 manipulation as a novel therapeutic strategy in gastric cancer, which still shows poor prognosis.
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19
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EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis. Cell Res 2015; 25:370-85. [PMID: 25656846 DOI: 10.1038/cr.2015.17] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2014] [Revised: 09/24/2014] [Accepted: 11/07/2014] [Indexed: 01/02/2023] Open
Abstract
In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 - molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.
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Inoue S, Kondo S, Parichy DM, Watanabe M. Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes. Pigment Cell Melanoma Res 2014; 27:190-200. [PMID: 24734316 DOI: 10.1111/pcmr.12192] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Skin pigment pattern formation in zebrafish requires pigment-cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N-glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.
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21
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Liang Y, Eng WS, Colquhoun DR, Dinglasan RR, Graham DR, Mahal LK. Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment. J Biol Chem 2014; 289:32526-37. [PMID: 25261472 DOI: 10.1074/jbc.m114.606269] [Citation(s) in RCA: 122] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.
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Affiliation(s)
- Yaxuan Liang
- From the Biomedical Chemistry Institute, Department of Chemistry, New York University, New York, New York 10003-6688
| | - William S Eng
- From the Biomedical Chemistry Institute, Department of Chemistry, New York University, New York, New York 10003-6688
| | - David R Colquhoun
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and
| | - Rhoel R Dinglasan
- W. Harry Feistone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
| | - David R Graham
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and
| | - Lara K Mahal
- From the Biomedical Chemistry Institute, Department of Chemistry, New York University, New York, New York 10003-6688,
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22
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Fénéant L, Levy S, Cocquerel L. CD81 and hepatitis C virus (HCV) infection. Viruses 2014; 6:535-72. [PMID: 24509809 PMCID: PMC3939471 DOI: 10.3390/v6020535] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Revised: 01/29/2014] [Accepted: 02/02/2014] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C Virus (HCV) infection is a global public health problem affecting over 160 million individuals worldwide. Its symptoms include chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped RNA virus mainly targeting liver cells and for which the initiation of infection occurs through a complex multistep process involving a series of specific cellular entry factors. This process is likely mediated through the formation of a tightly orchestrated complex of HCV entry factors at the plasma membrane. Among HCV entry factors, the tetraspanin CD81 is one of the best characterized and it is undoubtedly a key player in the HCV lifecycle. In this review, we detail the current knowledge on the involvement of CD81 in the HCV lifecycle, as well as in the immune response to HCV infection.
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Affiliation(s)
- Lucie Fénéant
- Center for Infection and Immunity of Lille, CNRS-UMR8204, Inserm-U1019, Institut Pasteur de Lille, Université Lille Nord de France, Institut de Biologie de Lille, 1 rue du Pr Calmette, CS50447, 59021 Lille Cedex, France.
| | - Shoshana Levy
- Department of Medicine, Division of Oncology, CCSR, Stanford University Medical Center, Stanford, CA 94305, USA.
| | - Laurence Cocquerel
- Center for Infection and Immunity of Lille, CNRS-UMR8204, Inserm-U1019, Institut Pasteur de Lille, Université Lille Nord de France, Institut de Biologie de Lille, 1 rue du Pr Calmette, CS50447, 59021 Lille Cedex, France.
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Abstract
An abundance of evidence shows supporting roles for tetraspanin proteins in human cancer. Many studies show that the expression of tetraspanins correlates with tumour stage, tumour type and patient outcome. In addition, perturbations of tetraspanins in tumour cell lines can considerably affect cell growth, morphology, invasion, tumour engraftment and metastasis. This Review emphasizes new studies that have used de novo mouse cancer models to show that select tetraspanin proteins have key roles in tumour initiation, promotion and metastasis. This Review also emphasizes how tetraspanin proteins can sometimes participate in tumour angiogenesis. These recent data build an increasingly strong case for tetraspanins as therapeutic targets.
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24
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Normal muscle regeneration requires tight control of muscle cell fusion by tetraspanins CD9 and CD81. Nat Commun 2013; 4:1674. [PMID: 23575678 DOI: 10.1038/ncomms2675] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2012] [Accepted: 03/01/2013] [Indexed: 12/16/2022] Open
Abstract
Skeletal muscle regeneration after injury follows a remarkable sequence of synchronized events. However, the mechanisms regulating the typical organization of the regenerating muscle at different stages remain largely unknown. Here we show that muscle regeneration in mice lacking either CD9 or CD81 is abnormal and characterized by the formation of discrete giant dystrophic myofibres, which form more quickly in the absence of both tetraspanins. We also show that, in myoblasts, these two tetraspanins associate with the immunoglobulin domain molecule CD9P-1 (EWI-F/FPRP), and that grafting of CD9P-1-depleted myoblasts in regenerating muscles also leads to abnormal regeneration. In vitro myotubes lacking CD9P-1 or both CD9 and CD81 fuse with a higher frequency than normal myotubes. Our study unveils a mechanism preventing inappropriate fusion of myotubes that has an important role in the restitution of normal muscle architecture during muscle regeneration.
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25
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Zhou B, Gibson-Corley KN, Herndon ME, Sun Y, Gustafson-Wagner E, Teoh-Fitzgerald M, Domann FE, Henry MD, Stipp CS. Integrin α3β1 can function to promote spontaneous metastasis and lung colonization of invasive breast carcinoma. Mol Cancer Res 2013; 12:143-154. [PMID: 24002891 DOI: 10.1158/1541-7786.mcr-13-0184] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
UNLABELLED Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1's contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed. IMPLICATIONS α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed.
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Affiliation(s)
- Bo Zhou
- Department of Biology, University of Iowa, Iowa City, IA, 52242 USA
| | | | - Mary E Herndon
- Department of Biology, University of Iowa, Iowa City, IA, 52242 USA
| | - Yihan Sun
- Department of Biology, University of Iowa, Iowa City, IA, 52242 USA
| | | | - Melissa Teoh-Fitzgerald
- Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA, 52242 USA
| | - Frederick E Domann
- Department of Pathology, University of Iowa, Iowa City, IA, 52242 USA.,Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA, 52242 USA.,Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, 52242 USA
| | - Michael D Henry
- Department of Pathology, University of Iowa, Iowa City, IA, 52242 USA.,Department of Molecular Physiology & Biophysics, University of Iowa, Iowa City, IA, 52242 USA.,Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, 52242 USA
| | - Christopher S Stipp
- Department of Biology, University of Iowa, Iowa City, IA, 52242 USA.,Department of Molecular Physiology & Biophysics, University of Iowa, Iowa City, IA, 52242 USA.,Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, 52242 USA
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26
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Gustafson-Wagner E, Stipp CS. The CD9/CD81 tetraspanin complex and tetraspanin CD151 regulate α3β1 integrin-dependent tumor cell behaviors by overlapping but distinct mechanisms. PLoS One 2013; 8:e61834. [PMID: 23613949 PMCID: PMC3629153 DOI: 10.1371/journal.pone.0061834] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2013] [Accepted: 03/15/2013] [Indexed: 01/16/2023] Open
Abstract
Integrin α3β1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3β1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3β1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3β1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3β1 association with other tetraspanins and impairs α3β1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3β1 functions is unclear. To begin to clarify which aspects of α3β1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3β1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3β1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3β1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3β1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3β1 integrin function. Importantly, some of CD9/CD81's α3β1 regulatory functions may not require CD9/CD81 to be physically linked to α3β1 by CD151.
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Affiliation(s)
| | - Christopher S. Stipp
- Department of Biology, University of Iowa, Iowa City, Iowa, United States of America
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa, United States of America
- Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa, United States of America
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27
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Prognostic significance of CD151 overexpression in non-small cell lung cancer. Lung Cancer 2013; 81:109-16. [PMID: 23570797 DOI: 10.1016/j.lungcan.2013.03.014] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2013] [Revised: 03/12/2013] [Accepted: 03/12/2013] [Indexed: 02/07/2023]
Abstract
The overexpression of tetraspanin CD151 - a transmembrane protein that promotes tumor invasion and metastasis - is associated with poor prognosis in various cancers. However, its clinical significance in non-small cell lung cancers (NSCLCs) has not been fully elucidated. We investigated CD151 expression status by immunohistochemical analysis in paraffin-embedded specimens obtained from 380 patients with surgically resected NSCLCs (245 squamous cell carcinomas [SCCs] and 135 adenocarcinomas [ADCs]) between 1994 and 2001. High CD151 expression was detected in 28.7% NSCLCs (20.8% of SCCs and 42.9% of ADCs) and was significantly associated with male gender, smokers, and ADCs. Moreover, elevated CD151 levels were correlated with reduced overall (OS) and disease-free survival (DFS), and were an independent negative prognostic factor for OS in NSCLC. According to histological type, high CD151 expression was an independent prognostic factor for lower OS in ADC, although not in each subtype, and the elevated CD151 expression levels were more common in solid-predominant tumors (48.3%). In contrast, there was no prognostic correlation in SCC. High CD151 expression appeared to correlate with aggressive behavior in NSCLC, suggesting that it may be a useful prognostic marker for lung ADC patients and a potential molecular target for NSCLC treatment.
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Schröder HM, Hoffmann SC, Hecker M, Korff T, Ludwig T. The tetraspanin network modulates MT1-MMP cell surface trafficking. Int J Biochem Cell Biol 2013; 45:1133-44. [PMID: 23500527 DOI: 10.1016/j.biocel.2013.02.020] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2012] [Revised: 02/11/2013] [Accepted: 02/21/2013] [Indexed: 10/27/2022]
Abstract
The membrane-type 1 matrix metalloproteinase (MT1-MMP) drives fundamental physiological and pathophysiological processes. Among other substrates, MT1-MMP cleaves components of the extracellular matrix and activates other matrix-cleaving proteases such as MMP-2. Trafficking is a highly effective means to modulate MT1-MMP cell surface expression, and hence regulate its function. Here, we describe the complex interaction of MT1-MMP with tetraspanins, their effects on MT1-MMP intracellular trafficking and proteolytic function. Tetraspanins are credited as membrane organizers that form a network within the membrane to regulate the trafficking of associated proteins. In short, we found MT1-MMP to interact with the tetraspanin-associated EWI-2 protein by a yeast two-hybrid screen. Immunoprecipitation analysis confirmed this interaction and further revealed that MT1-MMP also stably interacts with distinct tetraspanins (CD9, CD37, CD53, CD63, CD81, and CD82) and the tetraspanin-like MAL protein. By using different MT1-MMP truncation constructs and mutants, we observed that all tetraspanins and MAL associated with the hemopexin domain of MT1-MMP. Moreover, this interaction was independent of O-glycosylation of MT1-MMP and exclusively occurred in the endoplasmic reticulum. Here, the respective subcellular compartment was identified by fitting the MT1-MMP interaction pattern to a model for post-translational processing of MT1-MMP. In addition, tetraspanins differentially affected the cell surface localization of MT1-MMP, its capacity to activate pro-MMP-2, and the collagen invasion capacity. Interestingly, the degree of tetraspanin-MT1-MMP association did not correlate with its impact on MT1-MMP function. Tetraspanins thus distinctly affect MT1-MMP subcellular localization and function, and may constitute an effective mechanism to control MT1-MMP-dependent proteolysis at the cell surface.
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Affiliation(s)
- H M Schröder
- Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Heidelberg University, 69120 Heidelberg, Germany.
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29
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Potel J, Rassam P, Montpellier C, Kaestner L, Werkmeister E, Tews BA, Couturier C, Popescu CI, Baumert TF, Rubinstein E, Dubuisson J, Milhiet PE, Cocquerel L. EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry. Cell Microbiol 2013; 15:1234-52. [PMID: 23351194 PMCID: PMC7162402 DOI: 10.1111/cmi.12112] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Revised: 12/14/2012] [Accepted: 01/10/2013] [Indexed: 01/07/2023]
Abstract
CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin-enriched areas in the plasma membrane. In our study, we combined single-molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI-2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI-2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81 molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI-2wint promotes CD81 clustering and confinement in CD81-enriched areas. In addition, we showed that EWI-2wint influences the colocalization of CD81 with Claudin-1 - a co-receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI-2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.
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Affiliation(s)
- Julie Potel
- Hepatitis C Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS-UMR8204, Inserm-U1019, Pasteur Institute of Lille, Lille, France
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30
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Dornier E, Coumailleau F, Ottavi JF, Moretti J, Boucheix C, Mauduit P, Schweisguth F, Rubinstein E. TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals. ACTA ACUST UNITED AC 2012; 199:481-96. [PMID: 23091066 PMCID: PMC3483123 DOI: 10.1083/jcb.201201133] [Citation(s) in RCA: 145] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch activation. The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.
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Affiliation(s)
- Emmanuel Dornier
- Institut National de la Santé et de la Recherche Médicale, U1004, F-94807 Villejuif, France
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Yoon H, Song JM, Ryu CJ, Kim YG, Lee EK, Kang S, Kim SJ. An efficient strategy for cell-based antibody library selection using an integrated vector system. BMC Biotechnol 2012; 12:62. [PMID: 22989299 PMCID: PMC3505469 DOI: 10.1186/1472-6750-12-62] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2012] [Accepted: 09/13/2012] [Indexed: 12/19/2022] Open
Abstract
Background Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. Results A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. Conclusions This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.
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Affiliation(s)
- Hyerim Yoon
- Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejon 305-806, Republic of Korea
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Hong IK, Jeoung DI, Ha KS, Kim YM, Lee H. Tetraspanin CD151 stimulates adhesion-dependent activation of Ras, Rac, and Cdc42 by facilitating molecular association between β1 integrins and small GTPases. J Biol Chem 2012; 287:32027-39. [PMID: 22843693 DOI: 10.1074/jbc.m111.314443] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.
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Affiliation(s)
- In-Kee Hong
- Medical and Bio-Material Research Center, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701, Korea
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Gordón-Alonso M, Sala-Valdés M, Rocha-Perugini V, Pérez-Hernández D, López-Martín S, Ursa A, Alvarez S, Kolesnikova TV, Vázquez J, Sánchez-Madrid F, Yáñez-Mó M. EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection. THE JOURNAL OF IMMUNOLOGY 2012; 189:689-700. [PMID: 22689882 DOI: 10.4049/jimmunol.1103708] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.
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34
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IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses. Mol Cell Neurosci 2012; 50:238-49. [PMID: 22687584 DOI: 10.1016/j.mcn.2012.05.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2011] [Revised: 05/10/2012] [Accepted: 05/28/2012] [Indexed: 01/08/2023] Open
Abstract
Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axon terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.
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Tetraspanin-interacting protein IGSF8 is dispensable for mouse fertility. Fertil Steril 2012; 98:465-70. [PMID: 22609062 DOI: 10.1016/j.fertnstert.2012.04.029] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2012] [Revised: 04/13/2012] [Accepted: 04/17/2012] [Indexed: 12/27/2022]
Abstract
OBJECTIVE To determine the physiological role of IGSF8 for fertility. DESIGN Experimental prospective study. SETTING Academic basic research laboratory. ANIMAL(S) C57BL/6J and hybrid B6D2F1 mice, as well as Cd9 and Igsf8 knockout mice (C57BL/6J and 129/SvJ mix background), were used for this study. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) In vitro and in vivo fertility tests of Igsf8 knockout mice. RESULT(S) Tetraspanin family member CD9 plays an important role in sperm-egg fusion. Recently, some researchers have reported that CD9 tightly associates with the immunoglobulin superfamily member IGSF8 on the egg surface and that IGSF8 is undetectable on the surface of Cd9-deficient eggs. This led us to hypothesize that IGSF8 participates in sperm-egg fusion together with CD9. To examine the physiological role of IGSF8 in vivo, we generated Igsf8-deficient mice by homologous recombination and examined the fertility of the females. CONCLUSION(S) The Igsf8-deficient female mice showed no fertilization defect in vitro or in vivo. We observed that Igsf8-deficient eggs retained the normal level and localization of CD9, resulting in normal microvilli formation, which indicates that IGSF8 is dispensable in fertility.
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Conley SM, Stuck MW, Naash MI. Structural and functional relationships between photoreceptor tetraspanins and other superfamily members. Cell Mol Life Sci 2012; 69:1035-47. [PMID: 21655915 PMCID: PMC3224198 DOI: 10.1007/s00018-011-0736-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2011] [Revised: 05/12/2011] [Accepted: 05/16/2011] [Indexed: 12/14/2022]
Abstract
The two primary photoreceptor-specific tetraspanins are retinal degeneration slow (RDS) and rod outer segment membrane protein-1 (ROM-1). These proteins associate together to form different complexes necessary for the proper structure of the photoreceptor outer segment rim region. Mutations in RDS cause blinding retinal degenerative disease in both rods and cones by mechanisms that remain unknown. Tetraspanins are implicated in a variety of cellular processes and exert their function via the formation of tetraspanin-enriched microdomains. This review focuses on correlations between RDS and other members of the tetraspanin superfamily, particularly emphasizing protein structure, complex assembly, and post-translational modifications, with the goal of furthering our understanding of the structural and functional role of RDS and ROM-1 in outer segment morphogenesis and maintenance, and our understanding of the pathogenesis associated with RDS and ROM-1 mutations.
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Affiliation(s)
- Shannon M. Conley
- Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L Young Blvd., Oklahoma City, OK 73104 USA
| | - Michael W. Stuck
- Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L Young Blvd., Oklahoma City, OK 73104 USA
| | - Muna I. Naash
- Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L Young Blvd., Oklahoma City, OK 73104 USA
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Yang XH, Mirchev R, Deng X, Yacono P, Yang HL, Golan DE, Hemler ME. CD151 restricts the α6 integrin diffusion mode. J Cell Sci 2012; 125:1478-87. [PMID: 22328509 DOI: 10.1242/jcs.093963] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Laminin-binding integrins (α3β1, α6β1, α6β4, α7β1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA-MB-231 mammary cells, tetraspanin CD151 knockdown impairs α6 integrin clustering and functions without decreasing α6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of α6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of α6 integrin diffusion. In control cells α6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells α6 integrin shows mostly DMO. In control cells α6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell α6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote α6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and α6 RCD. For α6 integrins, physical association with CD151 promotes α6 RCD, in support of α6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. αv integrins), CD151 affects neither diffusion mode nor αv function. Hence, CD151 support of α6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.
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Affiliation(s)
- Xiuwei H Yang
- Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
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He B, Zhang YH, Richardson MM, Zhang JS, Rubinstein E, Zhang XA. Differential functions of phospholipid binding and palmitoylation of tumour suppressor EWI2/PGRL. Biochem J 2011; 437:399-411. [PMID: 21609323 PMCID: PMC4141501 DOI: 10.1042/bj20101381] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The tumour suppressor EWI2 associates with tetraspanins and regulates tumour cell movement and proliferation. The short cytoplasmic domain of EWI2 is positively charged; five out of the ten residues of this domain are basic. In the present study we demonstrated that the EWI2 cytoplasmic tail interacts specifically with negatively charged PIPs (phosphatidylinositol phosphates), but not with other membrane lipids. The PIPs that interact with EWI2 cytoplasmic tail include PtdIns5P, PtdIns4P, PtdIns3P, PtdIns(3,5)P(2) and PtdIns(3,4)P2. The binding affinity of PIPs to the EWI2 tail, however, is not solely based on charge because PtdIns5P, PtdIns4P and PtdIns3P have a higher affinity to EWI2 than PtdIns(3,5)P(2) and PtdIns(3,4)P(2) do. Mutation of either of two basic residue clusters in the EWI2 cytoplasmic tail abolishes PIP binding, and PIP binding is also determined by the position of basic residues in the EWI2 cytoplasmic tail. In addition, EWI2 is constitutively palmitoylated at the cytoplasmic cysteine residues located at the N-terminal of those basic residues. The PIP interaction is not required for, but appears to regulate, the palmitoylation, whereas palmitoylation is neither required for nor regulates the PIP interaction. Functionally, the PIP interaction regulates the stability of EWI2 proteins, whereas palmitoylation is needed for tetraspanin-EWI2 association and EWI2-dependent inhibition of cell migration and lamellipodia formation. For cell-cell adhesion and cell proliferation, the PIP interaction functions in opposition to the palmitoylation. In conclusion, the EWI2 cytoplasmic tail actively engages with the cell membrane via PIP binding and palmitoylation, which play differential roles in EWI2 functions.
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Affiliation(s)
- Bo He
- Vascular Biology Center, Center for Cancer Research, and Departments of Medicine and Molecular Science, University of Tennessee Health Science Center, Memphis, TN 38163, U.S.A
| | - Yanhui H. Zhang
- Vascular Biology Center, Center for Cancer Research, and Departments of Medicine and Molecular Science, University of Tennessee Health Science Center, Memphis, TN 38163, U.S.A
| | - Mekel M. Richardson
- Vascular Biology Center, Center for Cancer Research, and Departments of Medicine and Molecular Science, University of Tennessee Health Science Center, Memphis, TN 38163, U.S.A
| | | | - Eric Rubinstein
- Inserm, U1004, 14 Av Paul Vaillant Couturier, 94807, Villejuif, France and Univ. Paris Sud, Institut André Lwoff, 94807, Villejuif, France
| | - Xin A. Zhang
- Vascular Biology Center, Center for Cancer Research, and Departments of Medicine and Molecular Science, University of Tennessee Health Science Center, Memphis, TN 38163, U.S.A
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Wang HX, Kolesnikova TV, Denison C, Gygi SP, Hemler ME. The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization. J Cell Sci 2011; 124:2702-10. [PMID: 21771881 DOI: 10.1242/jcs.085449] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.
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Affiliation(s)
- Hong-Xing Wang
- Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
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Montpellier C, Tews BA, Poitrimole J, Rocha-Perugini V, D'Arienzo V, Potel J, Zhang XA, Rubinstein E, Dubuisson J, Cocquerel L. Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection. J Biol Chem 2011; 286:13954-65. [PMID: 21343309 DOI: 10.1074/jbc.m111.220103] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.
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Affiliation(s)
- Claire Montpellier
- Center for Infection and Immunity of Lille, Hepatitis C Laboratory, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, 59021 Lille, France
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Sadej R, Romanska H, Kavanagh D, Baldwin G, Takahashi T, Kalia N, Berditchevski F. Tetraspanin CD151 regulates transforming growth factor beta signaling: implication in tumor metastasis. Cancer Res 2010; 70:6059-70. [PMID: 20570898 DOI: 10.1158/0008-5472.can-09-3497] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Tetraspanin CD151 is associated with laminin-binding integrins and controls tumor cell migration and invasion. By analyzing responses of breast cancer cells to various growth factors, we showed that depletion of CD151 specifically attenuates transforming growth factor beta1 (TGFbeta1)-induced scattering and proliferation of breast cancer cells in three-dimensional Matrigel. CD151-dependent cell scattering requires its association with either alpha3beta1 or alpha6 integrins, but it is independent of the recruitment of CD151 to tetraspanin-enriched microdomains. We also found that CD151 regulates the compartmentalization of TGF-beta type I receptor (TbetaRI/ALK-5) and specifically controls the TGFbeta1-induced activation of p38. In contrast, signaling leading to activation of Smad2/3, c-Akt, and Erk1/2 proteins was comparable in CD151(+) and CD151(-) cells. Attenuation of TGFbeta1-induced responses correlated with reduced retention in the lung vascular bed, inhibition of pneumocyte-induced scattering of breast cancer cells in three-dimensional Matrigel, and decrease in experimental metastasis to the lungs. These results identify CD151 as a positive regulator of TGFbeta1-initiated signaling and highlight the important role played by this tetraspanin in TGFbeta1-induced breast cancer metastasis.
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Affiliation(s)
- Rafal Sadej
- Department of Pathology, School of Cancer Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom
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Chambrion C, Le Naour F. The tetraspanins CD9 and CD81 regulate CD9P1-induced effects on cell migration. PLoS One 2010; 5:e11219. [PMID: 20574531 PMCID: PMC2888588 DOI: 10.1371/journal.pone.0011219] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2010] [Accepted: 05/20/2010] [Indexed: 12/15/2022] Open
Abstract
CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a beta1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin alpha2beta1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility.
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Affiliation(s)
- Célia Chambrion
- Inserm U1004, Villejuif, France
- Univ. Paris-Sud 11, Institut André Lwoff, Villejuif, France
| | - François Le Naour
- Univ. Paris-Sud 11, Institut André Lwoff, Villejuif, France
- Inserm U785, Villejuif, France
- * E-mail:
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Abstract
Within the integrin family of cell adhesion receptors, integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 make up a laminin-binding subfamily. The literature is divided on the role of these laminin-binding integrins in metastasis, with different studies indicating either pro- or antimetastatic functions. The opposing roles of the laminin-binding integrins in different settings might derive in part from their unusually robust associations with tetraspanin proteins. Tetraspanins organise integrins into multiprotein complexes within discrete plasma membrane domains termed tetraspanin-enriched microdomains (TEMs). TEM association is crucial to the strikingly rapid cell migration mediated by some of the laminin-binding integrins. However, emerging data suggest that laminin-binding integrins also promote the stability of E-cadherin-based cell-cell junctions, and that tetraspanins are essential for this function as well. Thus, TEM association endows the laminin-binding integrins with both pro-invasive functions (rapid migration) and anti-invasive functions (stable cell junctions), and the composition of TEMs in different cell types might help determine the balance between these opposing activities. Unravelling the tetraspanin control mechanisms that regulate laminin-binding integrins will help to define the settings where inhibiting the function of these integrins would be helpful rather than harmful, and may create opportunities to modulate integrin activity in more sophisticated ways than simple functional blockade.
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Xu D, Sharma C, Hemler ME. Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein. FASEB J 2009; 23:3674-81. [PMID: 19587294 DOI: 10.1096/fj.09-133462] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Using mass spectrometry, we identified ADAM10 (a membrane-associated metalloproteinase) as a partner for TSPAN12, a tetraspanin protein. TSPAN12-ADAM10 interaction was confirmed by reciprocal coimmunoprecipitation in multiple tumor cell lines. TSPAN12, to a greater extent than other tetraspanins (CD81, CD151, CD9, and CD82), associated with ADAM10 but not with ADAM17. Overexpression of TSPAN12 enhanced ADAM10-dependent shedding of amyloid precursor protein (APP) in MCF7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. Conversely, siRNA ablation of endogenous TSPAN12 markedly diminished APP proteolysis in both cell lines. Furthermore, TSPAN12 overexpression enhanced ADAM10 prodomain maturation, whereas TSPAN12 ablation diminished ADAM10 maturation. A palmitoylation-deficient TSPAN12 mutant failed to associate with ADAM10, inhibited ADAM10-dependent proteolysis of APP, and inhibited ADAM10 maturation, most likely by interfering with endogenous wild-type TSPAN12. In conclusion, TSPAN12 serves as a novel and robust partner for ADAM10 and promotes ADAM10 maturation, thereby facilitating ADAM10-dependent proteolysis of APP. This novel mode of regulating APP cleavage is of relevance to Alzheimer's disease therapy.
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Affiliation(s)
- Daosong Xu
- Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA
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Myelin proteomics: molecular anatomy of an insulating sheath. Mol Neurobiol 2009; 40:55-72. [PMID: 19452287 PMCID: PMC2758371 DOI: 10.1007/s12035-009-8071-2] [Citation(s) in RCA: 226] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2009] [Accepted: 04/14/2009] [Indexed: 12/12/2022]
Abstract
Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies.
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Abstract
Despite high expression levels at the plasma membrane or in intracellular vesicles, tetraspanins remain among the most mysterious transmembrane molecules 20 years after their discovery. Several genetic studies in mammals and invertebrates have demonstrated key physiological roles for some of these tetraspanins, in particular in the immune response, sperm-egg fusion, photoreceptor function and the normal function of certain epithelia. Other studies have highlighted their ability to modulate cell migration and metastasis formation. Their role in the propagation of infectious agents has drawn recent attention, with evidence for HIV budding in tetraspanin-enriched plasma membrane domains. Infection of hepatocytic cells by two major pathogens, the hepatitis C virus and the malaria parasite, also requires the tetraspanin CD81. The function of tetraspanins is thought to be linked to their ability to associate with one another and a wealth of other integral proteins, thereby building up an interacting network or 'tetraspanin web'. On the basis of the biochemical dissection of the tetraspanin web and recent analysis of the dynamics of some of its constituents, we propose that tetraspanins tightly regulate transient interactions between a variety of molecules and as such favour the efficient assembly of specialized structures upon proper stimulation.
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Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo. Neoplasia 2009; 11:77-86, 4p following 86. [PMID: 19107234 DOI: 10.1593/neo.81180] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2008] [Revised: 10/22/2008] [Accepted: 10/23/2008] [Indexed: 01/28/2023] Open
Abstract
EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.
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48
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Lafleur MA, Xu D, Hemler ME. Tetraspanin proteins regulate membrane type-1 matrix metalloproteinase-dependent pericellular proteolysis. Mol Biol Cell 2009; 20:2030-40. [PMID: 19211836 DOI: 10.1091/mbc.e08-11-1149] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.
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Affiliation(s)
- Marc A Lafleur
- Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA
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49
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Hemler ME. Targeting of tetraspanin proteins--potential benefits and strategies. Nat Rev Drug Discov 2009; 7:747-58. [PMID: 18758472 DOI: 10.1038/nrd2659] [Citation(s) in RCA: 274] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The tetraspanin transmembrane proteins have emerged as key players in malignancy, the immune system, during fertilization and infectious disease processes. Tetraspanins engage in a wide range of specific molecular interactions, occurring through the formation of tetraspanin-enriched microdomains (TEMs). TEMs therefore serve as a starting point for understanding how tetraspanins affect cell signalling, adhesion, morphology, motility, fusion and virus infection. An abundance of recent evidence suggests that targeting tetraspanins, for example, by monoclonal antibodies, soluble large-loop proteins or RNAi technology, should be therapeutically beneficial.
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Affiliation(s)
- Martin E Hemler
- Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachussetts 02115, USA.
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50
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Vaisar T, Kassim SY, Gomez IG, Green PS, Hargarten S, Gough PJ, Parks WC, Wilson CL, Raines EW, Heinecke JW. MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages. Mol Cell Proteomics 2008; 8:1044-60. [PMID: 19116209 DOI: 10.1074/mcp.m800449-mcp200] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, beta(2) integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of beta(2) integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala(705) and Ile(706) of the beta(2) integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that beta(2) integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of beta(2) integrin.
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Affiliation(s)
- Tomás Vaisar
- Department of Medicine, School of Medicine, University of Washington, Seattle, Washington 98195, USA
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