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Cancers Jun 2023Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether...
Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether another Ca-binding protein, the CaM-related centrin1, binds to K-Ras and could mediate some K-Ras functions that were previously ascribed to CaM. While CaM and centrin1 appear to distinguish between peptides that were derived from their classical targets, they both bind to K-Ras in cells. Cellular BRET- and immunoprecipitation data suggest that CaM engages more with K-Ras than centrin1 and that the interaction with the C-terminal membrane anchor of K-Ras is sufficient for this. Surprisingly, binding of neither K-Ras nor its membrane anchor alone to CaM or centrin1 is sensitive to inhibition of prenylation. In support of an involvement of the G-domain of K-Ras in cellular complexes with these Ca-binding proteins, we find that oncogenic K-RasG12V displays increased engagement with both CaM and centrin1. This is abrogated by addition of the D38A effector-site mutation, suggesting that K-RasG12V is held together with CaM or centrin1 in complexes with effectors. When treated with CaM inhibitors, the BRET-interaction of K-RasG12V with centrin1 was also disrupted in the low micromolar range, comparable to that with CaM. While CaM predominates in regulating functional membrane anchorage of K-Ras, it has a very similar co-distribution with centrin1 on mitotic organelles. Given these results, a significant overlap of the CaM- and centrin1-dependent functions of K-Ras is suggested.
PubMed: 37370697
DOI: 10.3390/cancers15123087 -
Molecular & Cellular Proteomics : MCP Aug 2023Proteins 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...
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 (PALM2) 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 PALM2 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.
Topics: Humans; Cell Movement; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Proteomics
PubMed: 37328063
DOI: 10.1016/j.mcpro.2023.100593 -
International Journal of Molecular... Jun 2023Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic...
Among kidney cancers, clear cell renal cell carcinoma (ccRCC) has the highest incidence rate in adults. The survival rate of patients diagnosed as having metastatic ccRCC drastically declines even with intensive treatment. We examined the efficacy of simvastatin, a lipid-lowering drug with reduced mevalonate synthesis, in ccRCC treatment. Simvastatin was found to reduce cell viability and increase autophagy induction and apoptosis. In addition, it reduced cell metastasis and lipid accumulation, the target proteins of which can be reversed through mevalonate supplementation. Moreover, simvastatin suppressed cholesterol synthesis and protein prenylation that is essential for RhoA activation. Simvastatin might also reduce cancer metastasis by suppressing the RhoA pathway. A gene set enrichment analysis (GSEA) of the human ccRCC GSE53757 data set revealed that the RhoA and lipogenesis pathways are activated. In simvastatin-treated ccRCC cells, although RhoA was upregulated, it was mainly restrained in the cytosolic fraction and concomitantly reduced Rho-associated protein kinase activity. RhoA upregulation might be a negative feedback effect owing to the loss of RhoA activity caused by simvastatin, which can be restored by mevalonate. RhoA inactivation by simvastatin was correlated with decreased cell metastasis in the transwell assay, which was mimicked in dominantly negative RhoA-overexpressing cells. Thus, owing to the increased RhoA activation and cell metastasis in the human ccRCC dataset analysis, simvastatin-mediated Rho inactivation might serve as a therapeutic target for ccRCC patients. Altogether, simvastatin suppressed the cell viability and metastasis of ccRCC cells; thus, it is a potentially effective ccRCC adjunct therapy after clinical validation for ccRCC treatment.
Topics: Humans; Simvastatin; Carcinoma, Renal Cell; Mevalonic Acid; Kidney Neoplasms; Lipids; rhoA GTP-Binding Protein
PubMed: 37298689
DOI: 10.3390/ijms24119738 -
Nature Communications Jun 2023While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic...
While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic suppression of PGC1α defines an aggressive subset of chronic BRAF-inhibitor treated melanomas. A metabolism-centered pharmacological screen further identifies statins (HMGCR inhibitors) as a collateral vulnerability within PGC1α-suppressed BRAF-inhibitor resistant melanomas. Lower PGC1α levels mechanistically causes reduced RAB6B and RAB27A expression, whereby their combined re-expression reverses statin vulnerability. BRAF-inhibitor resistant cells with reduced PGC1α have increased integrin-FAK signaling and improved extracellular matrix detached survival cues that helps explain their increased metastatic ability. Statin treatment blocks cell growth by lowering RAB6B and RAB27A prenylation that reduces their membrane association and affects integrin localization and downstream signaling required for growth. These results suggest that chronic adaptation to BRAF-targeted treatments drive novel collateral metabolic vulnerabilities, and that HMGCR inhibitors may offer a strategy to treat melanomas recurring with suppressed PGC1α expression.
Topics: Humans; Proto-Oncogene Proteins B-raf; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Drug Collateral Sensitivity; Neoplasm Recurrence, Local; Melanoma; Protein Kinase Inhibitors; Integrins; Epigenesis, Genetic; Cell Line, Tumor; Mutation; Hydroxymethylglutaryl CoA Reductases
PubMed: 37277330
DOI: 10.1038/s41467-023-38968-7 -
Cold Spring Harbor Perspectives in... Jun 2023Choroideremia is an X-linked inherited retinal degeneration characterized by primary centripetal degeneration of the retinal pigment epithelium (RPE), with secondary...
Choroideremia is an X-linked inherited retinal degeneration characterized by primary centripetal degeneration of the retinal pigment epithelium (RPE), with secondary degeneration of the choroid and retina. Affected individuals experience reduced night vision in early adulthood with blindness in late middle age. The underlying gene encodes REP1, a protein involved in the prenylation of Rab GTPases essential for intracellular vesicle trafficking. Adeno-associated viral gene therapy has demonstrated some benefit in clinical trials for choroideremia. However, challenges remain in gaining regulatory approval. Choroideremia is slowly progressive, which presents difficulties in demonstrating benefit over short pivotal clinical trials that usually run for 1-2 years. Improvements in visual acuity are particularly challenging due to the initial negative effects of surgical detachment of the fovea. Despite these challenges, great progress toward a treatment has been made since choroideremia was first described in 1872.
PubMed: 37277205
DOI: 10.1101/cshperspect.a041279 -
Signal Transduction and Targeted Therapy May 2023The ever-increasing prevalence of noncommunicable diseases (NCDs) represents a major public health burden worldwide. The most common form of NCD is metabolic diseases,... (Review)
Review
The ever-increasing prevalence of noncommunicable diseases (NCDs) represents a major public health burden worldwide. The most common form of NCD is metabolic diseases, which affect people of all ages and usually manifest their pathobiology through life-threatening cardiovascular complications. A comprehensive understanding of the pathobiology of metabolic diseases will generate novel targets for improved therapies across the common metabolic spectrum. Protein posttranslational modification (PTM) is an important term that refers to biochemical modification of specific amino acid residues in target proteins, which immensely increases the functional diversity of the proteome. The range of PTMs includes phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, glycosylation, palmitoylation, myristoylation, prenylation, cholesterylation, glutathionylation, S-nitrosylation, sulfhydration, citrullination, ADP ribosylation, and several novel PTMs. Here, we offer a comprehensive review of PTMs and their roles in common metabolic diseases and pathological consequences, including diabetes, obesity, fatty liver diseases, hyperlipidemia, and atherosclerosis. Building upon this framework, we afford a through description of proteins and pathways involved in metabolic diseases by focusing on PTM-based protein modifications, showcase the pharmaceutical intervention of PTMs in preclinical studies and clinical trials, and offer future perspectives. Fundamental research defining the mechanisms whereby PTMs of proteins regulate metabolic diseases will open new avenues for therapeutic intervention.
Topics: Humans; Protein Processing, Post-Translational; Phosphorylation; Glycosylation; Proteome; Metabolic Diseases
PubMed: 37244925
DOI: 10.1038/s41392-023-01439-y -
Biomedicine & Pharmacotherapy =... Aug 2023Prenyltransferases (PTases) are known to play a role in embryonic development, normal tissue homeostasis and cancer by posttranslationally modifying proteins involved in... (Review)
Review
Prenyltransferases (PTases) are known to play a role in embryonic development, normal tissue homeostasis and cancer by posttranslationally modifying proteins involved in these processes. They are being discussed as potential drug targets in an increasing number of diseases, ranging from Alzheimer's disease to malaria. Protein prenylation and the development of specific PTase inhibitors (PTIs) have been subject to intense research in recent decades. Recently, the FDA approved lonafarnib, a specific farnesyltransferase inhibitor that acts directly on protein prenylation; and bempedoic acid, an ATP citrate lyase inhibitor that might alter intracellular isoprenoid composition, the relative concentrations of which can exert a decisive influence on protein prenylation. Both drugs represent the first approved agent in their respective substance class. Furthermore, an overwhelming number of processes and proteins that regulate protein prenylation have been identified over the years, many of which have been proposed as molecular targets for pharmacotherapy in their own right. However, certain aspects of protein prenylation, such as the regulation of PTase gene expression or the modulation of PTase activity by phosphorylation, have attracted less attention, despite their reported influence on tumor cell proliferation. Here, we want to summarize the advances regarding our understanding of the regulation of protein prenylation and the potential implications for drug development. Additionally, we want to suggest new lines of investigation that encompass the search for regulatory elements for PTases, especially at the genetic and epigenetic levels.
Topics: Protein Prenylation; Proteins; Dimethylallyltranstransferase; Enzyme Inhibitors; Terpenes; Prenylation
PubMed: 37236024
DOI: 10.1016/j.biopha.2023.114915 -
Cell Death and Differentiation Jul 2023SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also...
SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.
Topics: Mice; Animals; Humans; Mevalonic Acid; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Carcinoma, Squamous Cell; Ubiquitin Thiolesterase
PubMed: 37202505
DOI: 10.1038/s41418-023-01173-6 -
Nature Communications May 2023The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and...
The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.
Topics: Animals; Humans; Mice; Mice, Knockout; Neutrophils; Oxidoreductases; Proteins
PubMed: 37179332
DOI: 10.1038/s41467-023-38447-z -
Platelets Dec 2023Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase...
Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time ( < .005) and attenuated clot firmness ( < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV ( < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control ( < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.
Topics: Humans; Adenosine Diphosphate; Atorvastatin; Blood Platelets; Fibrinogen; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Prenylation; rab GTP-Binding Proteins; rap1 GTP-Binding Proteins; Thrombosis
PubMed: 37139869
DOI: 10.1080/09537104.2023.2206921