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Healy FM, Turner AL, Marensi V, MacEwan DJ. Mediating kinase activity in Ras-mutant cancer: potential for an individualised approach? Front Pharmacol 2024; 15:1441938. [PMID: 39372214 PMCID: PMC11450236 DOI: 10.3389/fphar.2024.1441938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2024] [Accepted: 09/06/2024] [Indexed: 10/08/2024] Open
Abstract
It is widely acknowledged that there is a considerable number of oncogenic mutations within the Ras superfamily of small GTPases which are the driving force behind a multitude of cancers. Ras proteins mediate a plethora of kinase pathways, including the MAPK, PI3K, and Ral pathways. Since Ras was considered undruggable until recently, pharmacological targeting of pathways downstream of Ras has been attempted to varying success, though drug resistance has often proven an issue. Nuances between kinase pathway activation in the presence of various Ras mutants are thought to contribute to the resistance, however, the reasoning behind activation of different pathways in different Ras mutational contexts is yet to be fully elucidated. Indeed, such disparities often depend on cancer type and disease progression. However, we are in a revolutionary age of Ras mutant targeted therapy, with direct-targeting KRAS-G12C inhibitors revolutionising the field and achieving FDA-approval in recent years. However, these are only beneficial in a subset of patients. Approximately 90% of Ras-mutant cancers are not KRAS-G12C mutant, and therefore raises the question as to whether other distinct amino acid substitutions within Ras may one day be targetable in a similar manner, and indeed whether better understanding of the downstream pathways these various mutants activate could further improve therapy. Here, we discuss the favouring of kinase pathways across an array of Ras-mutant oncogenic contexts and assess recent advances in pharmacological targeting of various Ras mutants. Ultimately, we will examine the utility of individualised pharmacological approaches to Ras-mediated cancer.
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Affiliation(s)
- Fiona M. Healy
- Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom
| | - Amy L. Turner
- Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom
| | - Vanessa Marensi
- Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom
- Chester Medical School, University of Chester, Chester, United Kingdom
| | - David J. MacEwan
- Department of Pharmacology and Therapeutics, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom
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Hildebrandt ER, Hussain SA, Sieburg MA, Ravishankar R, Asad N, Gore S, Ito T, Hougland JL, Dore TM, Schmidt WK. Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential. Bioorg Chem 2024; 147:107316. [PMID: 38583246 PMCID: PMC11098683 DOI: 10.1016/j.bioorg.2024.107316] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 02/16/2024] [Accepted: 03/26/2024] [Indexed: 04/09/2024]
Abstract
Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.
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Affiliation(s)
- Emily R Hildebrandt
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA
| | - Shaneela A Hussain
- New York University Abu Dhabi, Saadiyat Island, PO Box 129188, Abu Dhabi, UAE
| | | | - Rajani Ravishankar
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA
| | - Nadeem Asad
- New York University Abu Dhabi, Saadiyat Island, PO Box 129188, Abu Dhabi, UAE
| | - Sangram Gore
- New York University Abu Dhabi, Saadiyat Island, PO Box 129188, Abu Dhabi, UAE
| | - Takahiro Ito
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA
| | - James L Hougland
- Department of Chemistry, Syracuse University, Syracuse, NY, USA; Department of Biology, Syracuse University, Syracuse, NY, USA; BioInspired Syracuse, Syracuse University, Syracuse, NY, USA
| | - Timothy M Dore
- New York University Abu Dhabi, Saadiyat Island, PO Box 129188, Abu Dhabi, UAE; Department of Chemistry, University of Georgia, Athens, GA, USA
| | - Walter K Schmidt
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA.
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Mouhcine M, Kadil Y, Segmani I, Rahmoune I, Filali H. In silico Exploration of a Novel ICMT Inhibitor with More Solubility than Cysmethynil against Membrane Localization of KRAS Mutant in Colorectal Cancer. Curr Comput Aided Drug Des 2024; 20:1055-1069. [PMID: 38835128 DOI: 10.2174/0115734099264451231003172217] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2023] [Revised: 08/14/2023] [Accepted: 08/31/2023] [Indexed: 06/06/2024]
Abstract
BACKGROUND ICMT (isoprenylcysteine carboxyl methyltransferase) is an enzyme that plays a key role in the post-translational modification of the K-Ras protein. The carboxyl methylation of this protein by ICMT is important for its proper localization and function. Cysmethynil (2-[5-(3-methylphenyl)-l-octyl-lH-indolo-3-yl] acetamide) causes K-Ras mislocalization and interrupts pathways that control cancer cell growth and division through inhibition of ICMT, but its poor water solubility makes it difficult and impractical for clinical use. This indicates that relatively high amounts of cysmethynil would be required to achieve an effective dose, which could result in significant adverse effects in patients. OBJECTIVE The general objective of this work was to find virtually new compounds that present high solubility in water and are similar to the pharmacological activity of cysmethynil. MATERIALS AND METHODS Pharmacophore modeling, pharmacophore-based virtual screening, prediction of ADMET properties (absorption, distribution, metabolism, excretion, and toxicity), and water solubility were performed to recover a water-soluble molecule that shares the same chemical characteristics as cysmethynil using Discovery Studio v16.1.0 (DS16.1), SwissADME server, and pkCSM server. RESULTS In this study, ten pharmacophore model hypotheses were generated by exploiting the characteristics of cysmethynil. The pharmacophore model validated by the set test method was used to screen the "Elite Library®" and "Synergy Library" databases of Asinex. Only 1533 compounds corresponding to all the characteristics of the pharmacophore were retained. Then, the aqueous solubility in water at 25°C of these 1533 compounds was predicted by the Cheng and Merz model. Among these 1533 compounds, two had the optimal water solubility. Finally, the ADMET properties and Log S water solubility by three models (ESOL, Ali, and SILICOS-IT) of the two compounds and cysmethynil were compared, resulting in compound 2 as a potential inhibitor of ICMT. CONCLUSION According to the results obtained, the identified compound presented a high solubility in water and could be similar to the pharmacological activity of cysmethynil.
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Affiliation(s)
- Mohammed Mouhcine
- Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University of Casablanca, Casablanca, Morocco
| | - Youness Kadil
- Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University of Casablanca, Casablanca, Morocco
| | - Ibtihal Segmani
- Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University of Casablanca, Casablanca, Morocco
| | - Imane Rahmoune
- Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University of Casablanca, Casablanca, Morocco
| | - Houda Filali
- Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University of Casablanca, Casablanca, Morocco
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Deng DX, Li CY, Zheng ZY, Wen B, Liao LD, Zhang XJ, Li EM, Xu LY. Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin. Mol Cell Proteomics 2023; 22:100593. [PMID: 37328063 PMCID: PMC10393820 DOI: 10.1016/j.mcpro.2023.100593] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Revised: 05/28/2023] [Accepted: 06/12/2023] [Indexed: 06/18/2023] Open
Abstract
Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin's FERM domain and C408 in PALM2's CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.
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Affiliation(s)
- Dan-Xia Deng
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Institute of Oncologic Pathology, Shantou University Medical College, Shantou, Guangdong, China
| | - Cheng-Yu Li
- Department of Biochemistry and Molecular Biology, The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou, Guangdong, China; Guangdong Esophageal Cancer Research Institute, Shantou Sub-center, Cancer Research Cancer, Shantou University Medical College, Shantou, Guangdong, China
| | - Zhen-Yuan Zheng
- Department of Biochemistry and Molecular Biology, The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou, Guangdong, China; Guangdong Esophageal Cancer Research Institute, Shantou Sub-center, Cancer Research Cancer, Shantou University Medical College, Shantou, Guangdong, China
| | - Bing Wen
- Department of Biochemistry and Molecular Biology, The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou, Guangdong, China
| | - Lian-Di Liao
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Institute of Oncologic Pathology, Shantou University Medical College, Shantou, Guangdong, China
| | - Xiao-Jun Zhang
- Central Laboratory, Shantou University Medical College, Shantou, Guangdong, China
| | - En-Min Li
- Department of Biochemistry and Molecular Biology, The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Shantou University Medical College, Shantou, Guangdong, China.
| | - Li-Yan Xu
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Institute of Oncologic Pathology, Shantou University Medical College, Shantou, Guangdong, China; Guangdong Esophageal Cancer Research Institute, Shantou Sub-center, Cancer Research Cancer, Shantou University Medical College, Shantou, Guangdong, China.
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5
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Ma H, Wu F, Bai Y, Wang T, Ma S, Guo L, Liu G, Leng G, Kong Y, Zhang Y. Licoricidin combats gastric cancer by targeting the ICMT/Ras pathway in vitro and in vivo. Front Pharmacol 2022; 13:972825. [PMID: 36339587 PMCID: PMC9629146 DOI: 10.3389/fphar.2022.972825] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2022] [Accepted: 09/15/2022] [Indexed: 12/24/2022] Open
Abstract
Licoricidin, a type of isoflavonoid, is extracted from the root of Glycyrrhiza glabra. It has been widely proven that licoricidin possesses multiple biological activities, including anti-cancer effects and a powerful antimicrobial effect against Helicobacter pylori (H. pylori). However, the exact mechanism of licoricidin against gastric cancer remains unclear. In this study, we comprehensively explored the effects of licoricidin on MGC-803 gastric cancer cells in vitro and in vivo and further elucidated its mechanism of action. Our results revealed that licoricidin exhibited multiple anti-gastric cancer activities, including suppressing proliferation, inducing apoptosis, arresting the cell cycle in G0/G1 phase, and inhibiting the migration and invasion abilities of MGC-803 gastric cancer cells. In addition to this, a total of 5861 proteins were identified by quantitative proteomics research strategy of TMT labeling, of which 19 differential proteins (two upregulated and 17 downregulated) were screened out. Combining bioinformatics analyses and the reported roles in cancer progression of the 19 proteins, we speculated that isoprenyl carboxyl methyltransferase (ICMT) was the most likely target of licoricidin. Western blot assays and IHC assays subsequently proved that licoricidin significantly downregulated the expression of ICMT, both in MGC-803 cells and in xenograft tumors. Moreover, licoricidin effectively reduced the level of active Ras-GTP and blocked the phosphorylation of Raf and Erk, which may be involved in its anti-gastric cancer effects. In summary, we first demonstrated that licoricidin exerted favorable anti-gastric cancer activities via the ICMT/Ras pathway, which suggests that licoricidin, as a natural product, could be a novel candidate for the management of gastric cancer.
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Affiliation(s)
- Hanwei Ma
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
- Department of Pediatric Gastroenterology, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Fahong Wu
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Yinliang Bai
- Pharmacy Department, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Tianwei Wang
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Shangxian Ma
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Liuqing Guo
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Guiyuan Liu
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Guangxian Leng
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Yin Kong
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
| | - Youcheng Zhang
- Laboratory of Hepatic-Biliary-Pancreatic, Department of General Surgery, Lanzhou University Second Hospital, Lanzhou, Gansu, China
- *Correspondence: Youcheng Zhang,
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Haidar M, Jacquemin P. Past and Future Strategies to Inhibit Membrane Localization of the KRAS Oncogene. Int J Mol Sci 2021; 22:13193. [PMID: 34947990 PMCID: PMC8707736 DOI: 10.3390/ijms222413193] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Revised: 12/02/2021] [Accepted: 12/04/2021] [Indexed: 12/13/2022] Open
Abstract
KRAS is one of the most studied oncogenes. It is well known that KRAS undergoes post-translational modifications at its C-terminal end. These modifications are essential for its membrane location and activity. Despite significant efforts made in the past three decades to target the mechanisms involved in its membrane localization, no therapies have been approved and taken into the clinic. However, many studies have recently reintroduced interest in the development of KRAS inhibitors, either by directly targeting KRAS or indirectly through the inhibition of critical steps involved in post-translational KRAS modifications. In this review, we summarize the approaches that have been applied over the years to inhibit the membrane localization of KRAS in cancer and propose a new anti-KRAS strategy that could be used in clinic.
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Affiliation(s)
| | - Patrick Jacquemin
- De Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium;
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Functional Interplay between Methyltransferases and Inflammasomes in Inflammatory Responses and Diseases. Int J Mol Sci 2021; 22:ijms22147580. [PMID: 34299198 PMCID: PMC8306412 DOI: 10.3390/ijms22147580] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2021] [Revised: 07/07/2021] [Accepted: 07/13/2021] [Indexed: 02/08/2023] Open
Abstract
An inflammasome is an intracellular protein complex that is activated in response to a pathogenic infection and cellular damage. It triggers inflammatory responses by promoting inflammatory cell death (called pyroptosis) and the secretion of pro-inflammatory cytokines, interleukin (IL)-1β and IL-18. Many types of inflammasomes have been identified and demonstrated to play a central role in inducing inflammatory responses, leading to the onset and progression of numerous inflammatory diseases. Methylation is a biological process by which methyl groups are transferred from methyl donors to proteins, nucleic acids, and other cellular molecules. Methylation plays critical roles in various biological functions by modulating gene expression, protein activity, protein localization, and molecular stability, and aberrant regulation of methylation causes deleterious outcomes in various human diseases. Methylation is a key determinant of inflammatory responses and diseases. This review highlights the current understanding of the functional relationship between inflammasome regulation and methylation of cellular molecules in inflammatory responses and diseases.
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8
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Wang SR, Sun W, Zhou N, Zhao K, Li WJ, Chi ZP, Wang Y, Wang QM, Tong L, He ZX, Han HY, Chen ZG. Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma. HUA XI KOU QIANG YI XUE ZA ZHI = HUAXI KOUQIANG YIXUE ZAZHI = WEST CHINA JOURNAL OF STOMATOLOGY 2021; 39:64-73. [PMID: 33723939 DOI: 10.7518/hxkq.2021.01.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
OBJECTIVES This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC). METHODS Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry. RESULTS The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05). CONCLUSIONS Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.
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Affiliation(s)
- Shao-Ru Wang
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China.,School of Stomatology, Dalian Medical University, Dalian 116044, China
| | - Wei Sun
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
| | - Nan Zhou
- College of Stomatology, Weifang Medical University, Weifang 261021, China
| | - Kai Zhao
- School of Stomatology, Qingdao University, Qingdao 266003, China
| | - Wen-Jian Li
- School of Stomatology, Dalian Medical University, Dalian 116044, China
| | - Zeng-Peng Chi
- College of Stomatology, Weifang Medical University, Weifang 261021, China
| | - Ying Wang
- Dept. of Stomatology, Fourth People,s Hospital of Jinan, Jinan 250031, China
| | - Qi-Min Wang
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
| | - Lei Tong
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
| | - Zong-Xuan He
- Dept. of Oral and Maxillafacial Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266005, China
| | - Hong-Yu Han
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
| | - Zheng-Gang Chen
- Dept. of Stomatology, Qingdao Municipal Hospital, Qingdao University, Qingdao 266071, China
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9
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Ahearn IM, Court HR, Siddiqui F, Abankwa D, Philips MR. NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane. Life Sci Alliance 2021; 4:4/5/e202000972. [PMID: 33579760 PMCID: PMC7893820 DOI: 10.26508/lsa.202000972] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 01/26/2021] [Accepted: 01/27/2021] [Indexed: 12/17/2022] Open
Abstract
Among the RAS isoforms, NRAS uniquely requires carboxyl methylation by ICMT for delivery to the plasma membrane because of having only a single palmitoylation as a second targeting signal. Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.
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Affiliation(s)
- Ian M Ahearn
- The Ronald O Perelman Department of Dermatology, New York University Grossman School of Medicine, New York, NY, USA .,The Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY, USA.,Veterans Affairs New York Harbor Healthcare System, Manhattan Campus, New York, NY, USA
| | - Helen R Court
- The Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY, USA
| | - Farid Siddiqui
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
| | - Daniel Abankwa
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland.,Cancer Cell Biology and Drug Discovery Group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Mark R Philips
- The Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY, USA
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10
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Lamango NS, Nkembo AT, Ntantie E, Tawfeeq N. Polyisoprenylated Cysteinyl Amide Inhibitors: A Novel Approach to Controlling Cancers with Hyperactive Growth Signaling. Curr Med Chem 2021; 28:3476-3489. [PMID: 33176634 PMCID: PMC9175089 DOI: 10.2174/0929867327666201111140825] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2020] [Revised: 09/18/2020] [Accepted: 09/25/2020] [Indexed: 11/22/2022]
Abstract
Aberrant activation of monomeric G-protein signaling pathways drives some of the most aggressive cancers. Suppressing these hyperactivities has been the focus of efforts to obtain targeted therapies. Polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in various cancers. Its inhibition induces the death of cancer cells that harbor the constitutively active K-Ras proteins. Furthermore, the viability of cancer cells driven by factors upstream of K-Ras, such as overexpressed growth factors and their receptors or the mutationally-activated receptors, is also susceptible to PMPMEase inhibition. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were thus designed to target cancers with hyperactive signaling pathways involving the G-proteins. The PCAIs were, however, poor inhibitors of PMPMEase, with Ki values ranging from 3.7 to 20 μM. On the other hand, they inhibited cell viability, proliferation, colony formation, induced apoptosis in cells with mutant K-Ras and inhibited cell migration and invasion with EC50 values of 1 to 3 μM. HUVEC tube formation was inhibited at submicromolar concentrations through their disruption of actin filament organization. At the molecular level, the PCAIs at 2 to 5 μM depleted monomeric G-proteins such as K-Ras, RhoA, Cdc42 and Rac1. The PCAIs also deplete vinculin and fascin that are involved in actin organization and function while disrupting vinculin punctates in the process. These demonstrate a polyisoprenylation-dependent mechanism that explains the observed PCAIs' inhibition of the proliferative, invasive and angiogenic processes that promote both tumor growth and metastasis.
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Affiliation(s)
- Nazarius S. Lamango
- College of Pharmacy and Pharmaceutical Sciences Institute of Public Health, Florida A&M University, Tallahassee FL32307, USA
| | - Augustine T. Nkembo
- College of Pharmacy and Pharmaceutical Sciences Institute of Public Health, Florida A&M University, Tallahassee FL32307, USA
| | - Elizabeth Ntantie
- College of Pharmacy and Pharmaceutical Sciences Institute of Public Health, Florida A&M University, Tallahassee FL32307, USA
| | - Nada Tawfeeq
- College of Pharmacy and Pharmaceutical Sciences Institute of Public Health, Florida A&M University, Tallahassee FL32307, USA
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11
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Borini Etichetti CM, Arel Zalazar E, Cocordano N, Girardini J. Beyond the Mevalonate Pathway: Control of Post-Prenylation Processing by Mutant p53. Front Oncol 2020; 10:595034. [PMID: 33224889 PMCID: PMC7674641 DOI: 10.3389/fonc.2020.595034] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 10/08/2020] [Indexed: 12/21/2022] Open
Abstract
Missense mutations in the TP53 gene are among the most frequent alterations in human cancer. Consequently, many tumors show high expression of p53 point mutants, which may acquire novel activities that contribute to develop aggressive tumors. An unexpected aspect of mutant p53 function was uncovered by showing that some mutants can increase the malignant phenotype of tumor cells through alteration of the mevalonate pathway. Among metabolites generated through this pathway, isoprenoids are of particular interest, since they participate in a complex process of posttranslational modification known as prenylation. Recent evidence proposes that mutant p53 also enhances this process through transcriptional activation of ICMT, the gene encoding the methyl transferase responsible for the last step of protein prenylation. In this way, mutant p53 may act at different levels to promote prenylation of key proteins in tumorigenesis, including several members of the RAS and RHO families. Instead, wild type p53 acts in the opposite way, downregulating mevalonate pathway genes and ICMT. This oncogenic circuit also allows to establish potential connections with other metabolic pathways. The demand of acetyl-CoA for the mevalonate pathway may pose limitations in cell metabolism. Likewise, the dependence on S-adenosyl methionine for carboxymethylation, may expose cells to methionine stress. The involvement of protein prenylation in tumor progression offers a novel perspective to understand the antitumoral effects of mevalonate pathway inhibitors, such as statins, and to explore novel therapeutic strategies.
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Affiliation(s)
| | - Evelyn Arel Zalazar
- Instituto de Inmunología Clínica y Experimental de Rosario, IDICER, CONICET-UNR, Rosario, Argentina
| | - Nabila Cocordano
- Instituto de Inmunología Clínica y Experimental de Rosario, IDICER, CONICET-UNR, Rosario, Argentina
| | - Javier Girardini
- Instituto de Inmunología Clínica y Experimental de Rosario, IDICER, CONICET-UNR, Rosario, Argentina
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12
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Chai TF, Manu KA, Casey PJ, Wang M. Isoprenylcysteine carboxylmethyltransferase is required for the impact of mutant KRAS on TAZ protein level and cancer cell self-renewal. Oncogene 2020; 39:5373-5389. [PMID: 32561852 PMCID: PMC7391290 DOI: 10.1038/s41388-020-1364-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2020] [Revised: 05/29/2020] [Accepted: 06/08/2020] [Indexed: 12/28/2022]
Abstract
Cancer stem cells possess the capacity for self-renewal and resistance to chemotherapy. It is therefore crucial to understand the molecular regulators of stemness in the quest to develop effective cancer therapies. TAZ is a transcription activator that promotes stem cell functions in post-development mammalian cells; suppression of TAZ activity reduces or eliminates cancer stemness in select cancers. Isoprenylcysteine carboxylmethyltransferase (ICMT) is the unique enzyme of the last step of posttranslational prenylation processing pathway that modifies several oncogenic proteins, including RAS. We found that suppression of ICMT results in reduced self-renewal/stemness in KRAS-driven pancreatic and breast cancer cells. Silencing of ICMT led to significant reduction of TAZ protein levels and loss of self-renewal ability, which could be reversed by overexpressing mutant KRAS, demonstrating the functional impact of ICMT modification on the ability of KRAS to control TAZ stability and function. Contrary to expectation, YAP protein levels appear to be much less susceptible than TAZ to the regulation by ICMT and KRAS, and YAP is less consequential in regulating stemness characteristics in these cells. Further, we found that the ICMT-dependent KRAS regulation of TAZ was mediated through RAF, but not PI3K, signaling. Functionally, we demonstrate that a signaling cascade from ICMT modification of KRAS to TAZ protein stability supports cancer cell self-renewal abilities in both in vitro and in vivo settings. In addition, studies using the proof-of-concept small molecule inhibitors of ICMT confirmed its role in regulating TAZ and self-renewal, demonstrating the potential utility of targeting ICMT to control aggressive KRAS-driven cancers.
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Affiliation(s)
- Tin Fan Chai
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore.,Department of Biochemistry, National University of Singapore, Singapore, 117596, Singapore
| | - Kanjoormana Aryan Manu
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore
| | - Patrick J Casey
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore.,Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 27710, USA
| | - Mei Wang
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore. .,Department of Biochemistry, National University of Singapore, Singapore, 117596, Singapore.
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13
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Isoprenylcysteine Carboxyl Methyltransferase and Its Substrate Ras Are Critical Players Regulating TLR-Mediated Inflammatory Responses. Cells 2020; 9:cells9051216. [PMID: 32422978 PMCID: PMC7291029 DOI: 10.3390/cells9051216] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2020] [Revised: 05/03/2020] [Accepted: 05/11/2020] [Indexed: 12/12/2022] Open
Abstract
In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules.
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14
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Torres M, Rosselló CA, Fernández-García P, Lladó V, Kakhlon O, Escribá PV. The Implications for Cells of the Lipid Switches Driven by Protein-Membrane Interactions and the Development of Membrane Lipid Therapy. Int J Mol Sci 2020; 21:ijms21072322. [PMID: 32230887 PMCID: PMC7177374 DOI: 10.3390/ijms21072322] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 03/17/2020] [Accepted: 03/19/2020] [Indexed: 02/06/2023] Open
Abstract
The cell membrane contains a variety of receptors that interact with signaling molecules. However, agonist-receptor interactions not always activate a signaling cascade. Amphitropic membrane proteins are required for signal propagation upon ligand-induced receptor activation. These proteins localize to the plasma membrane or internal compartments; however, they are only activated by ligand-receptor complexes when both come into physical contact in membranes. These interactions enable signal propagation. Thus, signals may not propagate into the cell if peripheral proteins do not co-localize with receptors even in the presence of messengers. As the translocation of an amphitropic protein greatly depends on the membrane's lipid composition, regulation of the lipid bilayer emerges as a novel therapeutic strategy. Some of the signals controlled by proteins non-permanently bound to membranes produce dramatic changes in the cell's physiology. Indeed, changes in membrane lipids induce translocation of dozens of peripheral signaling proteins from or to the plasma membrane, which controls how cells behave. We called these changes "lipid switches", as they alter the cell's status (e.g., proliferation, differentiation, death, etc.) in response to the modulation of membrane lipids. Indeed, this discovery enables therapeutic interventions that modify the bilayer's lipids, an approach known as membrane-lipid therapy (MLT) or melitherapy.
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Affiliation(s)
- Manuel Torres
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa km 7.5, E-07122 Palma, Spain; (M.T.); (C.A.R.); (P.F.-G.); (V.L.)
- Department of R&D, Laminar Pharmaceuticals SL. ParcBit, Ed. Naorte B, E-07121 Palma, Spain
| | - Catalina Ana Rosselló
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa km 7.5, E-07122 Palma, Spain; (M.T.); (C.A.R.); (P.F.-G.); (V.L.)
- Department of R&D, Laminar Pharmaceuticals SL. ParcBit, Ed. Naorte B, E-07121 Palma, Spain
| | - Paula Fernández-García
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa km 7.5, E-07122 Palma, Spain; (M.T.); (C.A.R.); (P.F.-G.); (V.L.)
- Department of R&D, Laminar Pharmaceuticals SL. ParcBit, Ed. Naorte B, E-07121 Palma, Spain
| | - Victoria Lladó
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa km 7.5, E-07122 Palma, Spain; (M.T.); (C.A.R.); (P.F.-G.); (V.L.)
- Department of R&D, Laminar Pharmaceuticals SL. ParcBit, Ed. Naorte B, E-07121 Palma, Spain
| | - Or Kakhlon
- Department of Neurology, Hadassah-Hebrew University Medical Center, Ein Kerem, 91120 Jerusalem, Israel;
| | - Pablo Vicente Escribá
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa km 7.5, E-07122 Palma, Spain; (M.T.); (C.A.R.); (P.F.-G.); (V.L.)
- Correspondence:
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15
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Chen FY, Li X, Zhu HP, Huang W. Regulation of the Ras-Related Signaling Pathway by Small Molecules Containing an Indole Core Scaffold: A Potential Antitumor Therapy. Front Pharmacol 2020; 11:280. [PMID: 32231571 PMCID: PMC7082308 DOI: 10.3389/fphar.2020.00280] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2019] [Accepted: 02/27/2020] [Indexed: 12/29/2022] Open
Abstract
The Ras-Related signaling pathway plays an important role in cell development and differentiation. A growing body of evidence collected in recent years has shown that the aberrant activation of Ras is associated with tumor-related processes. Several studies have indicated that indole and its derivatives can target regulatory factors and interfere with or even block the aberrant Ras-Related pathway to treat or improve malignant tumors. In this review, we summarize the roles of indole and its derivatives in the isoprenylcysteine carboxyl methyltransferase-participant Ras membrane localization signaling pathway and Ras-GTP/Raf/MAPK signaling pathway through their regulatory mechanisms. Moreover, we briefly discuss the current treatment strategies that target these pathways. Our review will help guide the further study of the application of Ras-Related signaling pathway inhibitors.
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Affiliation(s)
- Fei-Yu Chen
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xiang Li
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Hong-Ping Zhu
- Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, China
| | - Wei Huang
- State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, China
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16
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Degirmenci U, Wang M, Hu J. Targeting Aberrant RAS/RAF/MEK/ERK Signaling for Cancer Therapy. Cells 2020; 9:E198. [PMID: 31941155 PMCID: PMC7017232 DOI: 10.3390/cells9010198] [Citation(s) in RCA: 366] [Impact Index Per Article: 73.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Revised: 12/29/2019] [Accepted: 01/10/2020] [Indexed: 12/13/2022] Open
Abstract
The RAS/RAF/MEK/ERK (MAPK) signaling cascade is essential for cell inter- and intra-cellular communication, which regulates fundamental cell functions such as growth, survival, and differentiation. The MAPK pathway also integrates signals from complex intracellular networks in performing cellular functions. Despite the initial discovery of the core elements of the MAPK pathways nearly four decades ago, additional findings continue to make a thorough understanding of the molecular mechanisms involved in the regulation of this pathway challenging. Considerable effort has been focused on the regulation of RAF, especially after the discovery of drug resistance and paradoxical activation upon inhibitor binding to the kinase. RAF activity is regulated by phosphorylation and conformation-dependent regulation, including auto-inhibition and dimerization. In this review, we summarize the recent major findings in the study of the RAS/RAF/MEK/ERK signaling cascade, particularly with respect to the impact on clinical cancer therapy.
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Affiliation(s)
- Ufuk Degirmenci
- Division of Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Crescent, Singapore 169610, Singapore
| | - Mei Wang
- Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore
| | - Jiancheng Hu
- Division of Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Crescent, Singapore 169610, Singapore
- Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore
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17
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Isoprenylcysteine carboxylmethyltransferase is associated with nasopharyngeal carcinoma chemoresistance and Ras activation. Biochem Biophys Res Commun 2019; 516:784-789. [PMID: 31253403 DOI: 10.1016/j.bbrc.2019.06.074] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2019] [Accepted: 06/15/2019] [Indexed: 12/29/2022]
Abstract
Development of chemo-resistance in nasopharyngeal carcinoma (NPC) poses the therapeutic challenge and its mechanisms are still poorly understood. In this work, we demonstrate that targeting isoprenylcysteine carboxylmethyltransferase (Icmt) is a therapeutic strategy to overcome NPC chemo-resistance. We found that Icmt mRNA and protein levels were increased in NPC cells after prolonged exposure to chemotherapy. Using pharmacological inhibitor cysmethynil or genetic siRNA approaches, we showed that Icmt inhibition was more effective against chemoresistant compared to chemosensitive NPC cells, suggesting that chemoresistant NPC cells is more dependent on Icmt function. The combination of Icmt inhibition with 5-FU or cisplatin resulted in greater efficacy than single chemotherapeutic agent alone in NPC. Notably, we demonstrated that the in vitro observations were translatable to in vivo NPC cancer xenograft mouse model. Mechanism analysis indicated that Icmt inhibition decreased Ras and RhoA activities, leading to the suppression of Ras and RhoA-mediated downstream signaling in NPC cells. The reverse of the inhibitory effects of cysmethynil by constitutively active Ras suggests that Ras is the critical effector of Icmt in NPC cells. Our work is the first to show that Icmt plays an important role in the development of NPC chemoresistance. Our findings also suggest that targeting Icmt represents a promising strategy to inhibit Ras function.
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18
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Zhou Y, Prakash P, Gorfe AA, Hancock JF. Ras and the Plasma Membrane: A Complicated Relationship. Cold Spring Harb Perspect Med 2018; 8:cshperspect.a031831. [PMID: 29229665 DOI: 10.1101/cshperspect.a031831] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The primary site of Ras signal transduction is the plasma membrane (PM). On the PM, the ubiquitously expressed Ras isoforms, H-, N-, and K-Ras, spatially segregate to nonoverlapping nanometer-sized domains, called nanoclusters, with further lateral segregation into nonoverlapping guanosine triphosphate (GTP)-bound and guanosine diphosphate (GDP)-bound nanoclusters. Effector binding and activation is restricted to GTP nanoclusters, rendering the underlying assembly mechanism essential to Ras signaling. Ras nanoclusters have distinct lipid compositions as a result of lipid-sorting specificity encoded in each Ras carboxy-terminal membrane anchor. The role of the G-domain in regulating anchor-membrane interactions is becoming clearer. Ras G-domains undergo significant conformational orientation changes on guanine nucleotide switch, leading to differential direct contacts between the G-domain and reorganization of the membrane anchor. Ras G-domains also contain weak dimer interfaces, resulting in homodimerization, which is an obligate step of nanoclustering. Modulating the formation of Ras dimers, the lipid composition of the PM or lateral dynamics of key PM phospholipids represent novel mechanisms whereby the extent of Ras nanoclustering can be regulated to tune the gain in Ras signaling circuits.
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Affiliation(s)
- Yong Zhou
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
| | - Priyanka Prakash
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
| | - Alemayehu A Gorfe
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
| | - John F Hancock
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030
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19
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Abstract
The high prevalence of KRAS mutations in human cancers and the lack of effective treatments for patients ranks KRAS among the most highly sought-after targets for preclinical oncologists. Pharmaceutical companies and academic laboratories have tried for decades to identify small molecule inhibitors of oncogenic KRAS proteins, but little progress has been made and many have labeled KRAS undruggable. However, recent progress in in silico screening, fragment-based drug design, disulfide tethered screening, and some emerging themes in RAS biology have caused the field to reconsider previously held notions about targeting KRAS. This review will cover some of the historical efforts to identify RAS inhibitors, and some of the most promising efforts currently being pursued.
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Affiliation(s)
- Matthew Holderfield
- NCI-Ras Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Frederick, Maryland 21702
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20
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Jeong A, Suazo KF, Wood WG, Distefano MD, Li L. Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer's disease. Crit Rev Biochem Mol Biol 2018; 53:279-310. [PMID: 29718780 PMCID: PMC6101676 DOI: 10.1080/10409238.2018.1458070] [Citation(s) in RCA: 103] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The mevalonate-isoprenoid-cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein-protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer's disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.
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Affiliation(s)
- Angela Jeong
- Departments of Experimental and Clinical Pharmacolog,University of Minnesota, Minneapolis, MN 55455
| | | | - W. Gibson Wood
- Departments of Pharmacology, University of Minnesota, Minneapolis, MN 55455
| | - Mark D. Distefano
- Departments of Chemistry,University of Minnesota, Minneapolis, MN 55455
| | - Ling Li
- Departments of Experimental and Clinical Pharmacolog,University of Minnesota, Minneapolis, MN 55455
- Departments of Pharmacology, University of Minnesota, Minneapolis, MN 55455
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21
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Pan Q, Liu R, Banu H, Ma L, Li H. Inhibition of isoprenylcysteine carboxylmethyltransferase sensitizes common chemotherapies in cervical cancer via Ras-dependent pathway. Biomed Pharmacother 2018; 99:169-175. [PMID: 29331763 DOI: 10.1016/j.biopha.2018.01.048] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Revised: 12/24/2017] [Accepted: 01/05/2018] [Indexed: 11/30/2022] Open
Abstract
Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last step of post-translational protein prenylation, which is essential for the stability and proper functions of many oncogenic proteins, such as Ras. Despite extensive studies on the roles of Icmt in tumor transformation and progression, little is known on the involvement ofIcmt in the development of tumor resistance to chemotherapy. Here we show the upregulation of Icmt as a persistent response to chemotherapy in cervical cancer cells. In-depth functional analysis demonstrated that Icmt inhibition significantly inhibited growth, induced apoptosis and augmented the inhibitory effects of chemotherapy drugs in cervical cancer in cell culture system and xenograft mouse model. Importantly, combination of Icmt specific inhibitor cysmethynil with doxorubicin or paclitaxel at sublethal concentration achieved almost full inhibition of tumor cell growth and survival. The remarkable synergy between chemotherapy drugs and Icmt inhibition in cervical cancer cells is likely due to the additional suppression of Ras and its downstream signaling pathways. We are the first to demonstrate the contribution of Icmt in tumor cells in response to chemotherapy. Our work also highlights Icmt inhibition as a sensitizing strategy for the treatment of cervical cancer or other Ras-driven tumors.
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Affiliation(s)
- Qin Pan
- Department of Obstetrics and Gynecology, Jingzhou Central Hospital, The Clinical Second Clinical Medical College of Yangtze University, Jingzhou, Hubei, China
| | - Rong Liu
- Department of Obstetrics and Gynecology, Jingzhou Central Hospital, The Clinical Second Clinical Medical College of Yangtze University, Jingzhou, Hubei, China
| | - Hasina Banu
- Department of Obstetrics and Gynecology, Jingzhou Central Hospital, The Clinical Second Clinical Medical College of Yangtze University, Jingzhou, Hubei, China
| | - Liang Ma
- Department of Orthopedics, Jingzhou Central Hospital, The Clinical Second Clinical Medical College of Yangtze University, Renmin Road 1, Jingzhou, Hubei, China.
| | - Hui Li
- Department of Obstetrics and Gynecology, Jingzhou Central Hospital, The Clinical Second Clinical Medical College of Yangtze University, Jingzhou, Hubei, China.
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22
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Lau HY, Wang M. Small change, big effect: Taking RAS by the tail through suppression of post-prenylation carboxylmethylation. Small GTPases 2017; 11:271-279. [PMID: 29261009 DOI: 10.1080/21541248.2017.1415637] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Mutant RAS isoforms are the most common oncogenes affecting human cancers. After decades of effort in developing drugs targeting oncogenic RAS-driven cancers, we are still charting an unclear path. Despite recent developments exemplified by KRAS (G12C) inhibitors, direct targeting of mutant RAS remains a difficult endeavor. Inhibiting RAS function by targeting its post-translational prenylation processing has remained an important approach, especially with recent progress on the study of isoprenylcysteine carboxylmethyltransferase (ICMT), the unique enzyme for the last step of prenylation processing of RAS isoforms and other substrates. Inhibition of ICMT has shown efficacy both in vitro and in vivo in RAS-mutant cancer models. We will discuss the roles of RAS family of proteins in human cancers and the impact of post-prenylation carboxylmethylation on RAS driven tumorigenesis. In addition, we will review what is known of the molecular and cellular impact of ICMT inhibition on cancer cells that underlie its anti-proliferative and pro-apoptosis efficacy.
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Affiliation(s)
- Hiu Yeung Lau
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School , Singapore
| | - Mei Wang
- Program in Cancer and Stem Cell Biology, Duke-NUS Medical School , Singapore.,Department of Biochemistry, National University of Singapore , Singapore
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23
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Shala-Lawrence A, Blanden MJ, Krylova SM, Gangopadhyay SA, Beloborodov SS, Hougland JL, Krylov SN. Simultaneous Analysis of a Non-Lipidated Protein and Its Lipidated Counterpart: Enabling Quantitative Investigation of Protein Lipidation’s Impact on Cellular Regulation. Anal Chem 2017; 89:13502-13507. [DOI: 10.1021/acs.analchem.7b03846] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Agnesa Shala-Lawrence
- Department
of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada
| | - Melanie J. Blanden
- Department
of Chemistry, Syracuse University, Syracuse, New York 13244, United States
| | - Svetlana M. Krylova
- Department
of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada
| | | | - Stanislav S. Beloborodov
- Department
of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada
| | - James L. Hougland
- Department
of Chemistry, Syracuse University, Syracuse, New York 13244, United States
| | - Sergey N. Krylov
- Department
of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada
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24
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Yang WS, Yeo SG, Yang S, Kim KH, Yoo BC, Cho JY. Isoprenyl carboxyl methyltransferase inhibitors: a brief review including recent patents. Amino Acids 2017; 49:1469-1485. [PMID: 28631011 PMCID: PMC5561173 DOI: 10.1007/s00726-017-2454-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2017] [Accepted: 06/14/2017] [Indexed: 12/03/2022]
Abstract
Among the enzymes involved in the post-translational modification of Ras, isoprenyl carboxyl methyltransferase (ICMT) has been explored by a number of researchers as a significant enzyme controlling the activation of Ras. Indeed, inhibition of ICMT exhibited promising anti-cancer activity against various cancer cell lines. This paper reviews patents and research articles published between 2009 and 2016 that reported inhibitors of ICMT as potential chemotherapeutic agents targeting Ras-induced growth factor signaling. Since ICMT inhibitors can modulate Ras signaling pathway, it might be possible to develop a new class of anti-cancer drugs targeting Ras-related cancers. Researchers have discovered indole-based small-molecular ICMT inhibitors through high-throughput screening. Researchers at Duke University identified a prototypical inhibitor, cysmethynil. At Singapore University, Ramanujulu and his colleagues patented more potent compounds by optimizing cysmethynil. In addition, Rodriguez and Stevenson at Universidad Complutense De Madrid and Cancer Therapeutics CRC PTY Ltd., respectively, have developed inhibitors based on formulas other than the indole base. However, further optimization of chemicals targeted to functional groups is needed to improve the characteristics of ICMT inhibitors related to their application as drugs, such as solubility, effectiveness, and safety, to facilitate clinical use.
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Affiliation(s)
- Woo Seok Yang
- Department of Genetic Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jang-gu, Suwon, 16419, Republic of Korea
| | - Seung-Gu Yeo
- Department of Radiation Oncology, Soonchunhyang University College of Medicine, Soonchunhyang University Hospital, Cheonan, 31151, Republic of Korea
| | - Sungjae Yang
- Department of Genetic Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jang-gu, Suwon, 16419, Republic of Korea
| | - Kyung-Hee Kim
- Biomarker Branch, Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea
| | - Byong Chul Yoo
- Biomarker Branch, Research Institute, National Cancer Center, Goyang, 10408, Republic of Korea.
| | - Jae Youl Cho
- Department of Genetic Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jang-gu, Suwon, 16419, Republic of Korea.
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Talele TT. Natural-Products-Inspired Use of the gem-Dimethyl Group in Medicinal Chemistry. J Med Chem 2017; 61:2166-2210. [DOI: 10.1021/acs.jmedchem.7b00315] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Affiliation(s)
- Tanaji T. Talele
- Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York 11439, United States
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Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function. J Neurosci 2017; 36:5107-14. [PMID: 27147662 DOI: 10.1523/jneurosci.0176-16.2016] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Accepted: 03/25/2016] [Indexed: 02/08/2023] Open
Abstract
UNLABELLED Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a "CAAX motif." We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α') with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α', and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific Rce1 knock-out mice, rod PDE6 in Icmt-deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the -AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of -AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the -AAX in PDE6 catalytic subunits in Rce1(-/-) mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.
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Do MT, Chai TF, Casey PJ, Wang M. Isoprenylcysteine carboxylmethyltransferase function is essential for RAB4A-mediated integrin β3 recycling, cell migration and cancer metastasis. Oncogene 2017; 36:5757-5767. [PMID: 28604748 PMCID: PMC5658678 DOI: 10.1038/onc.2017.183] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Revised: 04/25/2017] [Accepted: 05/04/2017] [Indexed: 12/27/2022]
Abstract
Isoprenylcysteine carboxylmethyltransferase (ICMT) catalyzes the post-translational modification of RAB GTPases that contain C-terminal CXC motifs. However, the functional impact of this modification on RAB proteins has not been actively explored. We found that inhibition of ICMT significantly reduced cell migration in vitro and cancer invasion and metastasis in vivo. This role of ICMT was found to be mediated by RAB4A, an essential regulator of the fast recycling of integrin β3. Integrin β3 regulates cell polarity and migration when localized appropriately to the plasma membrane, thereby having an essential role in cancer metastasis. ICMT catalyzed carboxylmethylation is critical for RAB4A activation and interaction with effectors, its localization to endosomes and recycling vesicles, and hence important for RAB4A-dependent integrin β3 recycling to plasma membrane. These findings bring attention to the effects of C-terminal carboxylmethylation on RAB GTPases and provide a rationale for targeting ICMT in the treatment of metastatic cancer.
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Affiliation(s)
- M T Do
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore
| | - T F Chai
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore
| | - P J Casey
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore.,Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA
| | - M Wang
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore.,Department of Biochemistry, National University of Singapore, Singapore
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Noguera-Salvà MA, Guardiola-Serrano F, Martin ML, Marcilla-Etxenike A, Bergo MO, Busquets X, Escribá PV. Role of the C-terminal basic amino acids and the lipid anchor of the Gγ 2 protein in membrane interactions and cell localization. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2017; 1859:1536-1547. [PMID: 28235469 DOI: 10.1016/j.bbamem.2017.02.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Revised: 02/16/2017] [Accepted: 02/18/2017] [Indexed: 12/25/2022]
Abstract
Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gβγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.
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Affiliation(s)
- Maria A Noguera-Salvà
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain
| | - Francisca Guardiola-Serrano
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain
| | - M Laura Martin
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain
| | - Amaia Marcilla-Etxenike
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain
| | - Martin O Bergo
- Sahlgrenska Cancer Center, University of Gothenburg, SE-405 30 Gothenburg, Sweden
| | - Xavier Busquets
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain.
| | - Pablo V Escribá
- Laboratory of Molecular Cell Biomedicine, Department of Biology, University of the Balearic Islands, E-07122 Palma, Balearic Islands, Spain
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The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting. Crit Rev Oncol Hematol 2017; 111:7-19. [PMID: 28259298 DOI: 10.1016/j.critrevonc.2017.01.002] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Revised: 11/15/2016] [Accepted: 01/05/2017] [Indexed: 01/17/2023] Open
Abstract
RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.
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Hildebrandt ER, Cheng M, Zhao P, Kim JH, Wells L, Schmidt WK. A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes. eLife 2016; 5. [PMID: 27525482 PMCID: PMC5014548 DOI: 10.7554/elife.15899] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Accepted: 08/14/2016] [Indexed: 11/21/2022] Open
Abstract
The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins. DOI:http://dx.doi.org/10.7554/eLife.15899.001
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Affiliation(s)
- Emily R Hildebrandt
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
| | - Michael Cheng
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
| | - Peng Zhao
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
| | - June H Kim
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
| | - Lance Wells
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
| | - Walter K Schmidt
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, United States
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Zhang F, Cheong JK. The renewed battle against RAS-mutant cancers. Cell Mol Life Sci 2016; 73:1845-58. [PMID: 26892781 PMCID: PMC11108322 DOI: 10.1007/s00018-016-2155-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2015] [Revised: 01/28/2016] [Accepted: 02/01/2016] [Indexed: 12/13/2022]
Abstract
The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients.
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Affiliation(s)
- Fuquan Zhang
- Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore
| | - Jit Kong Cheong
- Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, 8 College Road, Singapore, 169857, Singapore.
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32
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Butler KV, Bohn K, Hrycyna CA, Jin J. Non-Substrate Based, Small Molecule Inhibitors of the Human Isoprenylcysteine Carboxyl Methyltransferase. MEDCHEMCOMM 2016; 7:1016-1021. [PMID: 27547295 DOI: 10.1039/c6md00130k] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Activating mutations of human K-Ras proteins are among the most common oncogenic mutations, present in approximately 30% of all human cancers. Posttranslational modifications to K-Ras guide it to the plasma membrane and disruption of this localization inhibits the growth of Ras-driven cancers. The human isoprenylcysteine carboxyl methyltransferase (hIcmt) enzyme catalyzes the final α-carboxyl methylesterification of the C-terminal farnesyl cysteine of K-Ras, which is necessary for its proper localization. Thus, hIcmt inhibition is a regarded as a promising cancer therapy. A high quality inhibitor of hIcmt with in vivo activity would advance hIcmt research and drug development. Herein, Wwe report the results of a screen for small molecule hIcmt inhibitors in a library of molecules that were not hIcmt substrate analogs. The lead compound identified by this screen (1) was modified to remove chemical liabilities and to increase potency. The most potent resulting compound (5) inhibited hIcmt in vitro with low micromolar potency (IC50 = 1.5 ± 0.2 μM) and was kinetically characterized as a competitive inhibitor for prenylated substrates and a non-competitive inhibitor for the cofactor and methyl donor S-adenosylmethionine (SAM). These inhibitors offer important structure activity relationships for the future development of hIcmt inhibitors with in vivo activity.
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Affiliation(s)
- Kyle V Butler
- Departments of Structural and Chemical Biology, Oncological Sciences, and Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States
| | - Kelsey Bohn
- Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States
| | - Christine A Hrycyna
- Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States
| | - Jian Jin
- Departments of Structural and Chemical Biology, Oncological Sciences, and Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States
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Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis. mSphere 2016; 1:mSphere00084-15. [PMID: 27303728 PMCID: PMC4894686 DOI: 10.1128/msphere.00084-15] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2015] [Accepted: 03/03/2016] [Indexed: 12/01/2022] Open
Abstract
Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus. Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCECryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.
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8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells. Bioorg Med Chem 2015; 24:160-78. [PMID: 26706114 DOI: 10.1016/j.bmc.2015.11.043] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2015] [Revised: 11/23/2015] [Accepted: 11/29/2015] [Indexed: 01/05/2023]
Abstract
Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.
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35
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Flynn SC, Lindgren DE, Hougland JL. Quantitative determination of cellular farnesyltransferase activity: towards defining the minimum substrate reactivity for biologically relevant protein farnesylation. Chembiochem 2014; 15:2205-10. [PMID: 25182009 DOI: 10.1002/cbic.201402239] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2014] [Indexed: 11/10/2022]
Abstract
Prenylation is a post-translational modification wherein an isoprenoid group is attached to a protein substrate by a protein prenyltransferase. Hundreds of peptide sequences are in vitro substrates for protein farnesyltransferase (FTase), but it remains unknown which of these sequences can successfully compete for in vivo prenylation. Translating in vitro studies to predict in vivo protein farnesylation requires determining the minimum reactivity needed for modification by FTase within the cell. Towards this goal, we developed a reporter protein series spanning several orders of magnitude in FTase reactivity as a calibrated sensor for endogenous FTase activity. Our approach provides a minimally invasive method to monitor changes in cellular FTase activity in response to environmental or genetic factors. Determining the reactivity "threshold" for in vivo prenylation will help define the prenylated proteome and identify prenylation-dependent pathways for therapeutic targeting.
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Affiliation(s)
- Susan C Flynn
- Syracuse University Department of Chemistry, 1-014 Center for Science and Technology, Syracuse, NY 13244-4100 (USA)
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van Hattum H, Waldmann H. Chemical Biology Tools for Regulating RAS Signaling Complexity in Space and Time. ACTA ACUST UNITED AC 2014; 21:1185-95. [DOI: 10.1016/j.chembiol.2014.08.001] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2014] [Revised: 07/14/2014] [Accepted: 08/01/2014] [Indexed: 12/31/2022]
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Teh JT, Zhu WL, Ilkayeva OR, Li Y, Gooding J, Casey PJ, Summers SA, Newgard CB, Wang M. Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism. Oncogene 2014; 34:3296-304. [PMID: 25151967 DOI: 10.1038/onc.2014.260] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2014] [Revised: 06/09/2014] [Accepted: 07/03/2014] [Indexed: 12/20/2022]
Abstract
Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.
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Affiliation(s)
- J T Teh
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore
| | - W L Zhu
- 1] Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore [2] Department of Biochemistry, National University of Singapore, Singapore, Singapore
| | - O R Ilkayeva
- Sarah W Stedman Nutrition and Metabolism Center, and Duke Institute of Molecular Physiology, Duke University Medical Center, Durham, NC, USA
| | - Y Li
- Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore
| | - J Gooding
- Sarah W Stedman Nutrition and Metabolism Center, and Duke Institute of Molecular Physiology, Duke University Medical Center, Durham, NC, USA
| | - P J Casey
- Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore
| | - S A Summers
- Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore
| | - C B Newgard
- Sarah W Stedman Nutrition and Metabolism Center, and Duke Institute of Molecular Physiology, Duke University Medical Center, Durham, NC, USA
| | - M Wang
- 1] Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore [2] Department of Biochemistry, National University of Singapore, Singapore, Singapore
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Oleinik NV, Helke KL, Kistner-Griffin E, Krupenko NI, Krupenko SA. Rho GTPases RhoA and Rac1 mediate effects of dietary folate on metastatic potential of A549 cancer cells through the control of cofilin phosphorylation. J Biol Chem 2014; 289:26383-26394. [PMID: 25086046 DOI: 10.1074/jbc.m114.569657] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.
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Affiliation(s)
- Natalia V Oleinik
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Kristi L Helke
- Comparative Medicine and Laboratory Animal Resources, and Medical University of South Carolina, Charleston, South Carolina 29425
| | - Emily Kistner-Griffin
- Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Natalia I Krupenko
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425; Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425
| | - Sergey A Krupenko
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425; Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425.
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39
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Diver MM, Long SB. Mutational analysis of the integral membrane methyltransferase isoprenylcysteine carboxyl methyltransferase (ICMT) reveals potential substrate binding sites. J Biol Chem 2014; 289:26007-26020. [PMID: 25059662 DOI: 10.1074/jbc.m114.585125] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The eukaryotic integral membrane enzyme isoprenylcysteine carboxyl methyltransferase (ICMT) methylates the carboxylate of a lipid-modified cysteine at the C terminus of its protein substrates. This is the final post-translational modification of proteins containing a CAAX motif, including the oncoprotein Ras, and therefore, ICMT may serve as a therapeutic target in cancer development. ICMT has no discernible sequence homology with soluble methyltransferases, and aspects of its catalytic mechanism are unknown. For example, how both the methyl donor S-adenosyl-l-methionine (AdoMet), which is water-soluble, and the methyl acceptor isoprenylcysteine, which is lipophilic, are recognized within the same active site is not clear. To identify regions of ICMT critical for activity, we combined scanning mutagenesis with methyltransferase assays. We mutated nearly half of the residues of the ortholog of human ICMT from Anopheles gambiae and observed reduced or undetectable catalytic activity for 62 of the mutants. The crystal structure of a distantly related prokaryotic methyltransferase (Ma Mtase), which has sequence similarity with ICMT in its AdoMet binding site but methylates different substrates, provides context for the mutational analysis. The data suggest that ICMT and Ma MTase bind AdoMet in a similar manner. With regard to residues potentially involved in isoprenylcysteine binding, we identified numerous amino acids within transmembrane regions of ICMT that dramatically reduced catalytic activity when mutated. Certain substitutions of these caused substrate inhibition by isoprenylcysteine, suggesting that they contribute to the isoprenylcysteine binding site. The data provide evidence that the active site of ICMT spans both cytosolic and membrane-embedded regions of the protein.
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Affiliation(s)
- Melinda M Diver
- Structural Biology Program, Sloan-Kettering Institute and Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065; Graduate Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065
| | - Stephen B Long
- Structural Biology Program, Sloan-Kettering Institute and Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065.
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40
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Lau HY, Ramanujulu PM, Guo D, Yang T, Wirawan M, Casey PJ, Go ML, Wang M. An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo. Cancer Biol Ther 2014; 15:1280-91. [PMID: 24971579 DOI: 10.4161/cbt.29692] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Inhibitors of isoprenylcysteine carboxylmethyltransferase (Icmt) are promising anti-cancer agents, as modification by Icmt is an essential component of the protein prenylation pathway for a group of proteins that includes Ras GTPases. Cysmethynil, a prototypical indole-based inhibitor of Icmt, effectively inhibits tumor cell growth. However, the physical properties of cysmethynil, such as its low aqueous solubility, make it a poor candidate for clinical development. A novel amino-derivative of cysmethynil with superior physical properties and marked improvement in efficacy, termed compound 8.12, has recently been reported. We report here that Icmt (-/-) mouse embryonic fibroblasts (MEFs) are much more resistant to compound 8.12-induced cell death than their wild-type counterparts, providing evidence that the anti-proliferative effects of this compound are mediated through an Icmt specific mechanism. Treatment of PC3 prostate and HepG2 liver cancer cells with compound 8.12 resulted in pre-lamin A accumulation and Ras delocalization from the plasma membrane, both expected outcomes from inhibition of the Icmt-catalyzed carboxylmethylation. Treatment with compound 8.12 induced cell cycle arrest, autophagy and cell death, and abolished anchorage-independent colony formation. Consistent with its greater in vitro efficacy, compound 8.12 inhibited tumor growth with greater potency than cysmethynil in a xenograft mouse model. Further, a drug combination study identified synergistic antitumor efficacy of compound 8.12 and the epithelial growth factor receptor (EGFR)-inhibitor gefitinib, possibly through enhancement of autophagy. This study establishes compound 8.12 as a pharmacological inhibitor of Icmt that is an attractive candidate for further preclinical and clinical development.
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Affiliation(s)
- Hiu Yeung Lau
- Program of Cancer and Stem Cell Biology; Duke-NUS Graduate Medical School; Singapore
| | - Pondy M Ramanujulu
- Department of Pharmacy; Faculty of Science; National University of Singapore; Singapore
| | - Dianyan Guo
- Program of Cancer and Stem Cell Biology; Duke-NUS Graduate Medical School; Singapore
| | - Tianming Yang
- Department of Pharmacy; Faculty of Science; National University of Singapore; Singapore
| | - Melissa Wirawan
- Program of Cancer and Stem Cell Biology; Duke-NUS Graduate Medical School; Singapore
| | - Patrick J Casey
- Program of Cancer and Stem Cell Biology; Duke-NUS Graduate Medical School; Singapore
| | - Mei-Lin Go
- Department of Pharmacy; Faculty of Science; National University of Singapore; Singapore
| | - Mei Wang
- Program of Cancer and Stem Cell Biology; Duke-NUS Graduate Medical School; Singapore
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41
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Court H, Amoyel M, Hackman M, Lee KE, Xu R, Miller G, Bar-Sagi D, Bach EA, Bergö MO, Philips MR. Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression. J Clin Invest 2014; 123:4681-94. [PMID: 24216479 DOI: 10.1172/jci65764] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Accepted: 08/08/2013] [Indexed: 12/20/2022] Open
Abstract
RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.
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MESH Headings
- Animals
- Animals, Genetically Modified
- Carcinoma in Situ/genetics
- Carcinoma in Situ/metabolism
- Carcinoma in Situ/pathology
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/pathology
- Cell Line, Tumor
- Disease Models, Animal
- Drosophila melanogaster/genetics
- Drosophila melanogaster/growth & development
- Drosophila melanogaster/metabolism
- Female
- Genes, ras
- Humans
- Male
- Metaplasia
- Mice
- Mice, 129 Strain
- Mice, Inbred C57BL
- Mice, Knockout
- Mice, Mutant Strains
- Mice, Transgenic
- Mutation
- Pancreas/metabolism
- Pancreas/pathology
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/pathology
- Protein Methyltransferases/deficiency
- Protein Methyltransferases/genetics
- Receptor, Notch1/metabolism
- Signal Transduction
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42
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Montenegro MF, Sánchez-del-Campo L, Fernández-Pérez MP, Sáez-Ayala M, Cabezas-Herrera J, Rodríguez-López JN. Targeting the epigenetic machinery of cancer cells. Oncogene 2014; 34:135-43. [PMID: 24469033 DOI: 10.1038/onc.2013.605] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 12/20/2013] [Indexed: 02/07/2023]
Abstract
Cancer is characterized by uncontrolled cell growth and the acquisition of metastatic properties. In most cases, the activation of oncogenes and/or deactivation of tumour suppressor genes lead to uncontrolled cell cycle progression and inactivation of apoptotic mechanisms. Although the underlying mechanisms of carcinogenesis remain unknown, increasing evidence links aberrant regulation of methylation to tumourigenesis. In addition to the methylation of DNA and histones, methylation of nonhistone proteins, such as transcription factors, is also implicated in the biology and development of cancer. Because the metabolic cycling of methionine is a key pathway for many of these methylating reactions, strategies to target the epigenetic machinery of cancer cells could result in novel and efficient anticancer therapies. The application of these new epigenetic therapies could be of utility in the promotion of E2F1-dependent apoptosis in cancer cells, in avoiding metastatic pathways and/or in sensitizing tumour cells to radiotherapy.
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Affiliation(s)
- M F Montenegro
- Department of Biochemistry and Molecular Biology A, University of Murcia, Murcia, Spain
| | - L Sánchez-del-Campo
- Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK
| | - M P Fernández-Pérez
- Department of Biochemistry and Molecular Biology A, University of Murcia, Murcia, Spain
| | - M Sáez-Ayala
- Department of Biochemistry and Molecular Biology A, University of Murcia, Murcia, Spain
| | - J Cabezas-Herrera
- Translational Cancer Research Group, University Hospital Virgen de la Arrixaca (IMIB), Murcia, Spain
| | - J N Rodríguez-López
- Department of Biochemistry and Molecular Biology A, University of Murcia, Murcia, Spain
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43
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Gangopadhyay SA, Losito EL, Hougland JL. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition. Biochemistry 2014; 53:434-46. [PMID: 24344934 DOI: 10.1021/bi4011732] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.
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44
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Tamanoi F, Lu J. Recent progress in developing small molecule inhibitors designed to interfere with ras membrane association: toward inhibiting K-Ras and N-Ras functions. Enzymes 2013; 34 Pt. B:181-200. [PMID: 25034105 DOI: 10.1016/b978-0-12-420146-0.00008-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
K-Ras and N-Ras are mutated in a wide range of human cancers, thus making these proteins attractive targets of anticancer drug development. However, no effective compounds have been obtained so far. One of the approaches taken to inhibit the function of K-Ras and N-Ras is to interfere with their membrane association. Various attempts have been taken. In the first example, we examine the approach conceived in early 1990s to inhibit protein prenylation that is required for their membrane association. The initial premise that the inhibition of Ras farnesylation leads to the inhibition of Ras was not realized, mainly due to alternative prenylation of K-Ras and N-Ras proteins. This led to the idea that the combined inhibition of FTase and GGTase-I can block membrane association of K-Ras and N-Ras. Dual specificity inhibitors of FTase and GGTase-I (DPIs) were also developed. These compounds were tested in preclinical and clinical studies. It appears that sufficiently high concentration of the drug to inhibit K-Ras was not achieved in previous attempts. In addition, dose-limiting toxicity has been observed and this was primarily ascribed to GGTase-I inhibition. Strategies to confer cancer targeting capabilities to the inhibitors may overcome the dose-limiting toxicity. In the second approach, postprenylation events were exploited. This led to the development of various inhibitors including the ICMT inhibitors. Finally, recent identification of compounds that inhibit the interaction between K-Ras and PDE-δ is discussed.
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Affiliation(s)
- Fuyuhiko Tamanoi
- Department of Microbiology, Immunology & Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California, USA.
| | - Jie Lu
- Department of Microbiology, Immunology & Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California, USA
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45
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Activated Ras as a Therapeutic Target: Constraints on Directly Targeting Ras Isoforms and Wild-Type versus Mutated Proteins. ISRN ONCOLOGY 2013; 2013:536529. [PMID: 24294527 PMCID: PMC3833460 DOI: 10.1155/2013/536529] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 09/15/2013] [Accepted: 10/04/2013] [Indexed: 12/12/2022]
Abstract
The ability to selectively and directly target activated Ras would provide immense utility for treatment of the numerous cancers that are driven by oncogenic Ras mutations. Patients with disorders driven by overactivated wild-type Ras proteins, such as type 1 neurofibromatosis, might also benefit from progress made in that context. Activated Ras is an extremely challenging direct drug target due to the inherent difficulties in disrupting the protein:protein interactions that underlie its activation and function. Major investments have been made to target Ras through indirect routes. Inhibition of farnesyl transferase to block Ras maturation has failed in large clinical trials. Likely reasons for this disappointing outcome include the significant and underappreciated differences in the isoforms of Ras. It is still plausible that inhibition of farnesyl transferase will prove effective for disease that is driven by activated H-Ras. The principal current focus of drugs entering clinic trial is inhibition of pathways downstream of activated Ras, for example, trametinib, a first-in-class MEK inhibitor. The complexity of signaling that is driven by activated Ras indicates that effective inhibition of oncogenic transduction through this approach will be difficult, with resistance being likely to emerge through switch to parallel pathways. Durable disease responses will probably require combinatorial block of several downstream targets.
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46
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Stevenson GI, Yong S, Fechner GA, Neve J, Lock A, Avery VM. Solid-phase synthesis of Biotin-S-Farnesyl-L-Cysteine, a surrogate substrate for isoprenylcysteine Carboxylmethyltransferase (ICMT). Bioorg Med Chem Lett 2013; 23:5671-3. [PMID: 23988355 DOI: 10.1016/j.bmcl.2013.08.022] [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: 06/17/2013] [Revised: 07/29/2013] [Accepted: 08/05/2013] [Indexed: 10/26/2022]
Abstract
Inhibition of isoprenylcysteine Carboxylmethyltransferase (ICMT) is of particular interest as a potential target for the development of cancer chemotherapeutic agents. Screening for inhibitors of ICMT utilises a scintillation proximity assay (SPA) in which Biotin-S-Farnesyl-L-Cysteine (BFC) acts as a surrogate substrate. A solid-phase synthesis protocol for the preparation of BFC using 2-chlorotrityl chloride resin as a solid support has been developed to provide sufficient supply of BFC for high throughput screening (HTS) and subsequent chemistry campaigns to target inhibitors of ICMT. The BFC prepared by this method can be produced quickly on large scale and is stable when stored at -20 °C as a solid, in solution, or on the resin.
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Affiliation(s)
- Graeme I Stevenson
- Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland 4111, Australia.
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47
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Ibrahim MX, Sayin VI, Akula MK, Liu M, Fong LG, Young SG, Bergo MO. Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria. Science 2013; 340:1330-3. [PMID: 23686339 PMCID: PMC4295631 DOI: 10.1126/science.1238880] [Citation(s) in RCA: 94] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders.
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Affiliation(s)
- Mohamed X. Ibrahim
- Sahlgrenska Cancer Center, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, S-41390 Gothenburg, Sweden
| | - Volkan I. Sayin
- Sahlgrenska Cancer Center, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, S-41390 Gothenburg, Sweden
| | - Murali K. Akula
- Sahlgrenska Cancer Center, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, S-41390 Gothenburg, Sweden
| | - Meng Liu
- Sahlgrenska Cancer Center, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, S-41390 Gothenburg, Sweden
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, S-41345 Gothenburg, Sweden
| | - Loren G. Fong
- Departments of Medicine and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Stephen G. Young
- Departments of Medicine and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
| | - Martin O. Bergo
- Sahlgrenska Cancer Center, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, S-41390 Gothenburg, Sweden
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48
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Ivanov SS, Roy C. Host lipidation: a mechanism for spatial regulation of Legionella effectors. Curr Top Microbiol Immunol 2013; 376:135-54. [PMID: 23918175 DOI: 10.1007/82_2013_344] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/08/2022]
Abstract
Bacterial pathogens have evolved the capacity to translocate proteins into the cytosol of infected cells to manipulate host processes. How do pathogens regulate spatially these bacterial effector proteins once they are released into the host cell? One mechanism, which is used by Legionella and other bacterial pathogens, is to encode effectors that mimic the substrates of eukaryotic lipid transferases. In this review we discuss three membrane-targeting pathways in eukaryotes that are exploited by Legionella and other pathogens-prenylation, palmitoylation, and myristoylation. Lipidation of bacterial substrates primes the effectors for coincidence detection-mediated targeting onto membrane-bound organelles by increasing membrane affinity. Intracellular membrane-targeting strategies that exploit protein fatty acylation and prenylation direct bacterial effectors to compartments where their target substrates reside and thus are critical for effector function.
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Affiliation(s)
- Stanimir S Ivanov
- Department of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave, New Haven, CT, 06519, USA,
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49
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Hougland JL, Gangopadhyay SA, Fierke CA. Expansion of protein farnesyltransferase specificity using "tunable" active site interactions: development of bioengineered prenylation pathways. J Biol Chem 2012; 287:38090-100. [PMID: 22992747 DOI: 10.1074/jbc.m112.404954] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be "tuned" using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein.
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Affiliation(s)
- James L Hougland
- Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
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50
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Novelli G, D'Apice MR. Protein farnesylation and disease. J Inherit Metab Dis 2012; 35:917-26. [PMID: 22307208 DOI: 10.1007/s10545-011-9445-y] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2011] [Revised: 12/16/2011] [Accepted: 12/22/2011] [Indexed: 12/19/2022]
Abstract
Prenylation consists of the addition of an isoprenoid group to a cysteine residue located near the carboxyl terminal of a protein. This enzymatic posttranslational modification is important for the maturation and processing of proteins. Both processes are necessary to mediate protein-protein and membrane-protein associations, in addition to regulating the localisation and function of proteins. The severe phenotype of animals deficient in enzymes involved in both prenylation and maturation highlights the significance of these processes. Moreover, alterations in the genes coding for isoprenylated proteins or enzymes that are involved in both prenylation and maturation processes have been found to be the basis of severe human diseases, such as cancer, neurodegenerative disorders, retinitis pigmentosa, and premature ageing syndromes. Recent studies on isoprenylation and postprenylation processing in pathological conditions have unveiled surprising aspects of these modifications and their roles in different cellular pathways. The identification of these enzymes as therapeutic targets has led researchers to validate their effects in vitro and in vivo as antitumour or antiageing agents. This review attempts to summarise the basic aspects of protein isoprenylation and postprenylation, integrating our data with that observed in other studies to provide a comprehensive scenario of progeroid syndromes and the therapeutic avenues.
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Affiliation(s)
- Giuseppe Novelli
- Department of Biopathology and Diagnostic Imaging, University of Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.
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