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Pharmacological Research Dec 2022Ras-GAP SH3 domain binding proteins (G3BPs) are a family of RNA-binding proteins that include G3BP1 and G3BP2 in mammals. The protein structure of G3BP2 allows it to... (Review)
Review
Ras-GAP SH3 domain binding proteins (G3BPs) are a family of RNA-binding proteins that include G3BP1 and G3BP2 in mammals. The protein structure of G3BP2 allows it to bind with RNA or protein and regulate the nucleoplasmic shuttle, therefore participating in a variety of biological functions such as cell growth, differentiation and migration, and RNA and protein metabolism. G3BP2 is abnormally expressed in many tumors and diseases, such as breast cancer, lung cancer, prostate cancer, cardiac hypertrophy, and atherosclerosis. Many researchers have found that G3BP2 may be a potential therapeutic target. In this review, we examine the structure and expression regulation of G3BP2, and further summarize the function of G3BP2 from three aspects: RNA stabilization, protein subcellular localization, and stress granules assembling to provide a broader understanding of the role of G3BP2.
Topics: Male; Animals; Humans; RNA Recognition Motif Proteins; Poly-ADP-Ribose Binding Proteins; DNA Helicases; RNA Helicases; Carrier Proteins; Breast Neoplasms; RNA; Mammals; RNA-Binding Proteins; Adaptor Proteins, Signal Transducing
PubMed: 36336216
DOI: 10.1016/j.phrs.2022.106548 -
Autophagy Jul 2023Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular...
Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular stress. SG formation and elimination process is a conserved cellular strategy to promote cell survival, although the precise regulation of this process is poorly understood. Here, we screened six E3 ubiquitin ligases present in SGs and identified TRIM21 (tripartite motif containing 21) as a central regulator of SG homeostasis that is highly enriched in SGs of cells under arsenite-induced oxidative stress. Knockdown of promotes SG formation whereas overexpression of inhibits the formation of physiological and pathological SGs associated with neurodegenerative diseases. TRIM21 catalyzes K63-linked ubiquitination of the SG core protein, G3BP1 (G3BP stress granule assembly factor 1), and G3BP1 ubiquitination can effectively inhibit LLPS, . Recent reports suggested the involvement of macroautophagy/autophagy, as a stress response pathway, in the regulation of SG homeostasis. We systematically investigated well-defined autophagy receptors and identified SQSTM1/p62 (sequestosome 1) and CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) as the primary receptors that directly interact with G3BP1 during arsenite-induced stress. Endogenous SQSTM1 and CALCOCO2 localize to the periphery of SGs under oxidative stress and mediate SG elimination, as single knockout of each receptor causes accumulation of physiological and pathological SGs. Collectively, our study broadens the understanding in the regulation of SG homeostasis by showing that TRIM21 and autophagy receptors modulate SG formation and elimination respectively, suggesting the possibility of clinical targeting of these molecules in therapeutic strategies for neurodegenerative diseases. ACTB: actin beta; ALS: amyotrophic lateral sclerosis; BafA1: bafilomycin A; BECN1: beclin 1; C9orf72: C9orf72-SMCR8 complex subunit; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Co-IP: co-immunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; G3BP1: G3BP stress granule assembly factor 1; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; NES: nuclear export signal; OPTN: optineurin; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; SG: stress granule; TAX1BP1: Tax1 binding protein 1; TOLLIP: toll interacting protein; TRIM21: tripartite motif containing 21; TRIM56: tripartite motif containing 56; UB: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.
Topics: Sequestosome-1 Protein; DNA Helicases; Arsenites; Stress Granules; C9orf72 Protein; Calcium; Autophagy; RNA Helicases; RNA Recognition Motif Proteins; Poly-ADP-Ribose Binding Proteins; Ubiquitination; Carrier Proteins; Apoptosis Regulatory Proteins; Homeostasis; Ubiquitins
PubMed: 36692217
DOI: 10.1080/15548627.2022.2164427 -
Redox Biology May 2022A cytosolic iron chaperone poly(rC)-binding protein 1 (PCBP1) is a multifunctional RNA-binding protein involving gene transcription, RNA regulation, and iron loading to...
A cytosolic iron chaperone poly(rC)-binding protein 1 (PCBP1) is a multifunctional RNA-binding protein involving gene transcription, RNA regulation, and iron loading to ferritins. PCBP1 is also known to repress autophagy, but the role of PCBP1 in ferritinophagy and ferroptosis remains unrevealed. Therefore, we examined the role of PCBP1 in ferritinophagy-mediated ferroptosis in head and neck cancer (HNC) cells. The effects of system xc cystine/glutamate antiporter (xCT) inhibitors and PCBP1 gene silencing/overexpression were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed by assessing cell viability and death, lipid reactive oxygen species and iron production, lipid, malondialdehyde, mRNA/protein expression, and autophagy flux assays. Interaction between PCBP1 and BECN1 mRNA was also examined by luciferase and RNA-protein pull-down assays. PCBP1 gene silencing increased autophagosome generation and autophagic flux. Conversely, PCBP1 upregulation inhibited autophagy activation via direct binding to the CU-rich elements on the 3'-untranslated region (3'-UTR) of BECN1 mRNA. The internal deletion or mutation of the 3'-UTR F2 region recovered BECN1 mRNA stability repressed by PCBP1, resulting in enhanced ferritinophagy-mediated ferroptosis. Besides, PCBP1 knockdown promoted polyunsaturated fatty acid peroxidation by increasing ALOX15 expression. Further, excess iron accumulation caused mitochondrial dysfunction in PCBP1-suppressed cells. A ferroptosis inducer sulfasalazine significantly suppressed tumor growth in mice with the transplantation of PCBP1-silenced HNC. Our data suggest that the dual functions of PCBP1 repressing BECN1 and ALOX15 mRNAs contribute to attenuating cancer susceptibility to ferroptosis inducers.
Topics: Animals; Arachidonate 15-Lipoxygenase; Autophagy; Beclin-1; Carrier Proteins; DNA-Binding Proteins; Ferroptosis; Head and Neck Neoplasms; Humans; Iron; Lipids; Mice; RNA; RNA, Messenger; RNA-Binding Proteins
PubMed: 35290903
DOI: 10.1016/j.redox.2022.102276 -
Redox Biology Oct 2022Guanosine triphosphate binding protein 4 (GTPBP4) is a key regulator of cell cycle progression and MAPK activation. However, how its biological properties intersect with...
Guanosine triphosphate binding protein 4 (GTPBP4) is a key regulator of cell cycle progression and MAPK activation. However, how its biological properties intersect with cellular metabolism in hepatocellular carcinoma (HCC) development remains poorly unexplained. Here, high GTPBP4 expression is found to be significantly associated with worse clinical outcomes in patients with HCC. Moreover, GTPBP4 upregulation is paralleled by DNA promoter hypomethylation and regulated by DNMT3A, a DNA methyltransferase. Additionally, both gain- and loss-of-function studies demonstrate that GTPBP4 promotes HCC growth and metastasis in vitro and in vivo. Mechanically, GTPBP4 can induce dimeric pyruvate kinase M2 (PKM2) formation through protein sumoylation modification to promote aerobic glycolysis in HCC. Notably, active GTPBP4 facilitates SUMO1 protein activation by UBA2, and acts as a linker bridging activated SUMO1 protein and PKM2 protein to induce PKM2 sumoylation. Furthermore, SUMO-modified PKM2 relocates from the cytoplasm to the nucleus may also could contribute to HCC progression through activating epithelial-mesenchymal transition (EMT) and STAT3 signaling pathway. Shikonin, a PKM2-specific inhibitor, can attenuate PKM2 dependent HCC glycolytic reprogramming, growth and metastasis promoted by GTPBP4, which offers a promising therapeutic candidate for HCC patients. Our findings indicate that GTPBP4-PKM2 regulatory axis plays a vital role in promoting HCC proliferation as well as metastasis by aerobic glycolysis and offer a promising therapeutic target for HCC patients.
Topics: Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; DNA; GTP-Binding Proteins; Gene Expression Regulation, Neoplastic; Glucose; Glycolysis; Humans; Liver Neoplasms; Methyltransferases; Nuclear Proteins; Pyruvate Kinase; Ubiquitin-Activating Enzymes
PubMed: 36116159
DOI: 10.1016/j.redox.2022.102458 -
Journal of Cachexia, Sarcopenia and... Dec 2022Sarcopenia is defined as an age-related progressive loss of muscle mass and/or strength. Although different factors can contribute to this disease, the underlying...
BACKGROUND
Sarcopenia is defined as an age-related progressive loss of muscle mass and/or strength. Although different factors can contribute to this disease, the underlying mechanisms remain unclear. We assessed transcriptional heterogeneity in skeletal muscles from sarcopenic and control mice at single-cell resolution.
METHODS
A mouse model was established to study sarcopenic skeletal muscles. Single-cell RNA-seq was performed on tibialis anterior (TA) muscle cells collected from sarcopenic and control mice. A series of bioinformatic analyses were carried out to identify and compare different cell types under different conditions. Immunofluorescence staining and western blotting were used to validate the findings from single-cell experiments. Tube formation assays were conducted to further evaluate the effects of Gbp2 on endothelial cells during angiogenesis.
RESULTS
A murine sarcopenia model was successfully established using a senescence-accelerated mouse strain (SAMP6, n = 5). Sarcopenia phenotype was induced by administration of dexamethasone (20 mg/kg) and reduced physical activity. Senescence-resistant mice strain (SAMR1) and SAMP6 strain with similar activity but injected with PBS were recruited as two control groups. As signs of sarcopenia, body weight, muscle cell counts and cross-sectional fibre area were all significantly decreased in sarcopenic mice (P value = 0.004, 0.03 and 0.035, respectively). After quality control, 13 612 TA muscle single-cell transcriptomes were retained for analysis. Fourteen cell clusters were identified from the profiled cells. Among them, two distinct endothelial subtypes were found to be dominant in the sarcopenia group (42.2% cells) and in the two control groups (59.1% and 47.9% cells), respectively. 191 differentially expressed genes were detected between the two endothelial subtypes. Sarcopenia-specific endothelial cell subtype exhibited a dramatic increase in the interferon family genes and the interferon-inducible guanylate-binding protein (GBP) family gene expressions. For example, Igtp and Gbp2 in sarcopenic endothelial cells were 5.4 and 13.3 times higher than those in the control groups, respectively. We further validated our findings in muscle specimens of sarcopenia patients and observed that GBP2 levels were increased in endothelial cells of a subset of patients (11 of 40 patients, 27.5%), and we identified significantly higher CD31 and GBP2 co-localization (P value = 0.001128). Finally, we overexpressed Gbp2 in human umbilical vein endothelial cells in vitro. The endothelial cells with elevated Gbp2 expression displayed compromised tube formation.
CONCLUSIONS
Our single-cell-based results suggested that endothelial cells may play critical roles in sarcopenia development through interferon-GBP signalling pathways, highlighting new therapeutic directions to slow down or even reverse age-related sarcopenia.
Topics: Humans; Mice; Animals; Interferons; Cross-Sectional Studies; Carrier Proteins; Endothelial Cells; Single-Cell Gene Expression Analysis; Sarcopenia; Muscle, Skeletal
PubMed: 36162807
DOI: 10.1002/jcsm.13091 -
JCI Insight Jan 2020Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in...
Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in premature termination codons (PTCs) that cause RNA degradation and a reduction of MyBP-C in HCM patient hearts. However, a reduction in MyBP-C has not been consistently observed in MYBPC3-mutant induced pluripotent stem cell cardiomyocytes (iPSCMs). To determine early MYBPC3 mutation effects, we used patient and genome-engineered iPSCMs. iPSCMs with frameshift mutations were compared with iPSCMs with MYBPC3 promoter and translational start site deletions, revealing that allelic loss of function is the primary inciting consequence of mutations causing PTCs. Despite a reduction in wild-type mRNA in all heterozygous iPSCMs, no reduction in MyBP-C protein was observed, indicating protein-level compensation through what we believe is a previously uncharacterized mechanism. Although homozygous mutant iPSCMs exhibited contractile dysregulation, heterozygous mutant iPSCMs had normal contractile function in the context of compensated MyBP-C levels. Agnostic RNA-Seq analysis revealed differential expression in genes involved in protein folding as the only dysregulated gene set. To determine how MYBPC3-mutant iPSCMs achieve compensated MyBP-C levels, sarcomeric protein synthesis and degradation were measured with stable isotope labeling. Heterozygous mutant iPSCMs showed reduced MyBP-C synthesis rates but a slower rate of MyBP-C degradation. These findings indicate that cardiomyocytes have an innate capacity to attain normal MyBP-C stoichiometry despite MYBPC3 allelic loss of function due to truncating mutations. Modulating MyBP-C degradation to maintain MyBP-C protein levels may be a novel treatment approach upstream of contractile dysfunction for HCM.
Topics: Alleles; Cardiomyopathy, Hypertrophic; Carrier Proteins; Cell Line; Codon, Nonsense; Frameshift Mutation; Gene Editing; Genetic Predisposition to Disease; Heterozygote; Humans; Muscle Development; Mutation; Myocytes, Cardiac; RNA, Messenger; Sarcomeres; Transcriptome
PubMed: 31877118
DOI: 10.1172/jci.insight.133782 -
Cancer Cell Sep 2021Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has...
Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.
Topics: Bone Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Knockout Techniques; HEK293 Cells; Humans; Microfilament Proteins; Nerve Tissue Proteins; Oncogene Proteins, Fusion; Protein Stability; Proteolysis; Proto-Oncogene Protein c-fli-1; RNA-Binding Protein EWS; Sarcoma, Ewing; Trans-Activators
PubMed: 34329586
DOI: 10.1016/j.ccell.2021.07.003 -
Redox Biology Sep 2023Vascular endothelial cells (ECs) senescence plays a crucial role in vascular aging that promotes the initiation and progression of cardiovascular disease. The mutation...
Vascular endothelial cells (ECs) senescence plays a crucial role in vascular aging that promotes the initiation and progression of cardiovascular disease. The mutation of Grb10-interacting GYF protein 2 (GIGYF2) is strongly associated with the pathogenesis of aging-related diseases, whereas its role in regulating ECs senescence and dysfunction still remains elusive. In this study, we found aberrant hyperexpression of GIGYF2 in senescent human ECs and aortas of old mice. Silencing GIGYF2 in senescent ECs suppressed eNOS-uncoupling, senescence, and endothelial dysfunction. Conversely, in nonsenescent cells, overexpressing GIGYF2 promoted eNOS-uncoupling, cellular senescence, endothelial dysfunction, and activation of the mTORC1-SK61 pathway, which were ablated by rapamycin or antioxidant N-Acetyl-l-cysteine (NAC). Transcriptome analysis revealed that staufen double-stranded RNA binding protein 1 (STAU1) is remarkably downregulated in the GIGYF2-depleted ECs. STAU1 depletion significantly attenuated GIGYF2-induced cellular senescence, dysfunction, and inflammation in young ECs. Furthermore, we disclosed that GIGYF2 acting as an RNA binding protein (RBP) enhances STAU1 mRNA stability, and that the intron region of the late endosomal/lysosomal adaptor MAPK and mTOR activator 4 (LAMTOR4) could bind to STAU1 protein to upregulate LAMTOR4 expression. Immunofluorescence staining showed that GIGYF2 overexpression promoted the translocation of mTORC1 to lysosome. In the mice model, GIGYF2 Cdh-Cre mice protected aged mice from aging-associated vascular endothelium-dependent relaxation and arterial stiffness. Our work discloses that GIGYF2 serving as an RBP enhances the mRNA stability of STAU1 that upregulates LAMTOR4 expression through binding with its intron region, which activates the mTORC1-S6K1 signaling via recruitment of mTORC1 to the lysosomal membrane, ultimately leading to ECs senescence, dysfunction, and vascular aging. Disrupting the GIGYF2-STAU1-mTORC1 signaling cascade may represent a promising therapeutic approach against vascular aging and aging-related cardiovascular diseases.
Topics: Animals; Humans; Mice; Aging; Carrier Proteins; Cellular Senescence; Cytoskeletal Proteins; Endothelial Cells; Guanine Nucleotide Exchange Factors; Mechanistic Target of Rapamycin Complex 1; RNA-Binding Proteins
PubMed: 37517320
DOI: 10.1016/j.redox.2023.102824 -
RNA Biology Feb 2021The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP)... (Review)
Review
The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.
Topics: Animals; Autoantigens; Binding Sites; Carrier Proteins; Gene Expression Regulation; Humans; Multigene Family; Protein Binding; Protein Interaction Domains and Motifs; RNA; RNA Cleavage; RNA-Binding Proteins; Ribonucleoproteins; Signal Transduction; Substrate Specificity; SS-B Antigen
PubMed: 32233986
DOI: 10.1080/15476286.2020.1733787 -
Autophagy Sep 2021In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between...
In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in . Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an -null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.
Topics: Arabidopsis; Arabidopsis Proteins; Autophagy; Carrier Proteins; Gene Expression Regulation, Plant; HSP90 Heat-Shock Proteins; Heat-Shock Response; Macroautophagy; Proteomics; Tacrolimus Binding Proteins; Tandem Mass Spectrometry
PubMed: 32967551
DOI: 10.1080/15548627.2020.1820778