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1.
Cell Death Dis ; 15(2): 115, 2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38326336

RESUMO

Gasdermin D (GSDMD) functions as a pivotal executor of pyroptosis, eliciting cytokine secretion following cleavage by inflammatory caspases. However, the role of posttranslational modifications (PTMs) in GSDMD-mediated pyroptosis remains largely unexplored. In this study, we demonstrate that GSDMD can undergo acetylation at the Lysine 248 residue, and this acetylation enhances pyroptosis. We identify histone deacetylase 4 (HDAC4) as the specific deacetylase responsible for mediating GSDMD deacetylation, leading to the inhibition of pyroptosis both in vitro and in vivo. Deacetylation of GSDMD impairs its ubiquitination, resulting in the inhibition of pyroptosis. Intriguingly, phosphorylation of HDAC4 emerges as a critical regulatory mechanism promoting its ability to deacetylate GSDMD and suppress GSDMD-mediated pyroptosis. Additionally, we implicate Protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ) in the dephosphorylation of HDAC4, thereby nullifying its deacetylase activity on GSDMD. This study reveals a complex regulatory network involving HDAC4, PP1, and GSDMD. These findings provide valuable insights into the interplay among acetylation, ubiquitination, and phosphorylation in the regulation of pyroptosis, offering potential targets for further investigation in the field of inflammatory cell death.


Assuntos
Gasderminas , Histona Desacetilases , Proteína Fosfatase 1 , Piroptose , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Animais , Camundongos , Gasderminas/metabolismo
2.
Viruses ; 16(2)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38399988

RESUMO

Viruses evolve many strategies to ensure the efficient synthesis of their proteins. One such strategy is the inhibition of the integrated stress response-the mechanism through which infected cells arrest translation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). We have recently shown that the human common cold betacoronavirus OC43 actively inhibits eIF2α phosphorylation in response to sodium arsenite, a potent inducer of oxidative stress. In this work, we examined the modulation of integrated stress responses by OC43 and demonstrated that the negative feedback regulator of eIF2α phosphorylation GADD34 is strongly induced in infected cells. However, the upregulation of GADD34 expression induced by OC43 was independent from the activation of the integrated stress response and was not required for the inhibition of eIF2α phosphorylation in virus-infected cells. Our work reveals a complex interplay between the common cold coronavirus and the integrated stress response, in which efficient viral protein synthesis is ensured by the inhibition of eIF2α phosphorylation but the GADD34 negative feedback loop is disrupted.


Assuntos
Betacoronavirus , Resfriado Comum , Humanos , Betacoronavirus/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismo , Fosforilação , Biossíntese de Proteínas , Fator de Iniciação 2 em Eucariotos/metabolismo , eIF-2 Quinase/genética
3.
Mol Cancer ; 23(1): 34, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38360682

RESUMO

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive. METHODS: The differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa. RESULTS: CircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway. CONCLUSIONS: Our data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Camundongos , Humanos , Carcinoma de Células Renais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Circular/genética , Camundongos Nus , Hibridização in Situ Fluorescente , Linhagem Celular Tumoral , Transdução de Sinais/genética , Neoplasias Renais/genética , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células/genética , Peptídeos/genética , Regulação Neoplásica da Expressão Gênica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
4.
Cell Death Dis ; 15(2): 149, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365764

RESUMO

Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the homeostatic threshold, they become toxic to cells. In our study, we demonstrated that elesclomol, as a carrier of copper ions, caused an upregulation of protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), which plays a role in regulating substrate selectivity of protein phosphatase 1 during cuproptosis. Mechanistically, we investigated that PPP1R15A activated translation initiation by dephosphorylating eukaryotic translation initiation factor 2 subunit alpha at the S51 residue through protein phosphatase 1 and phosphorylating eukaryotic translation initiation factor 4E binding protein 1 at the T70 residue. In addition, PPP1R15A reduced H3K4 methylation by altering the phosphorylation of histone methyltransferases, which led to the silencing of MYC and G2M phase arrest.


Assuntos
Cobre , Neoplasias , Proteína Fosfatase 1 , Humanos , Cobre/metabolismo , Íons/metabolismo , Neoplasias/genética , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Apoptose/genética , Iniciação Traducional da Cadeia Peptídica/genética
5.
Methods Mol Biol ; 2740: 37-61, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38393468

RESUMO

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO). RIPPOs are tagged with the auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules, and PP1 substrate identification is conducted by SILAC proteomic-based approaches. Proteins in close proximity to RIPPOs are first identified through mass spectrometry (MS) analyses using the APEX2 system; then a list of differentially phosphorylated proteins upon RIPPOs rapid degradation (achieved via the AID system) is compiled via SILAC phospho-mass spectrometry. The "in silico" overlap between the two proteomes will be enriched for PP1 putative substrates. Several methods including fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), and in vitro assays can be used as substrate validations approaches.


Assuntos
Proteômica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Fosforilação , Ciclo Celular , Linhagem Celular , Holoenzimas/química , Holoenzimas/metabolismo
6.
Clin. transl. oncol. (Print) ; 26(1): 119-135, jan. 2024.
Artigo em Inglês | IBECS | ID: ibc-229151

RESUMO

Background Protein phosphatase 1 regulatory subunit 14B (PPP1R14B) is an oncogenic gene found in a variety of tumors, but its role in the prognosis and development of kidney renal clear cell carcinoma (KIRC) remains unknown. Our study aimed to determine whether PPP1R14B could be a prognostic biomarker for KIRC and its role in the development of KIRC. Methods In this work, we used The Cancer Genome Atlas (TCGA) database to explore the expression of PPP1R14B in tumor tissues, its relationship with the prognosis of tumor patients, and its role in tumor occurrence and development. We validated our findings using the International Cancer Genome Consortium (ICGC) cohort, our clinical samples, and in vitro experiments. Results PPP1R14B was upregulated in KIRC compared to adjacent normal tissue. Moreover, multivariate analysis revealed that upregulated PPP1R14B expression was an independent risk factor for KIRC progression. High-PPP1R14B groups had shorter overall survival (OS) and disease-free survival (DFS) in TCGA and ICGC cohorts. We used Cell Counting Kit-8 (CCK8) and scratch wound healing assay to explore the proliferation and migration of KIRC cells following PPP1R14B knockdown. Our results indicated that PPP1R14B knockdown significantly reduced the proliferation and migration of KIRC cells in vitro. We also explored the possible cellular mechanisms of PPP1R14B through the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene ontology (GO) analysis, and TISIDB analysis. The function enrich analysis revealed that PPP1R14B-related genes were mainly enriched in purine metabolism and the macromolecule catabolic process. PPP1R14B expression was associated with tumor-infiltrating immune cells (TIICs) in the TCGA cohort, and the results of single-cell RNA-seq (scRNA) further demonstrated that PPP1R14B expression was associated with the enhanced infiltration of CD8 + T lymphocytes (AU)


Assuntos
Humanos , Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Proteína Fosfatase 1/sangue , Prognóstico
7.
Acta Pharmacol Sin ; 45(4): 790-802, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38191913

RESUMO

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 µM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis. This study revealed that PPP1R15A expression was increased in osteoporosis in both human and mice. Inhibition of PPP1R15A by specific knockdown or an allosteric inhibitor Sephin1 mitigated murine osteoclast formation in vitro and attenuated ovariectomy-induced osteoporosis in vivo. PPP1R15A inhibition also suppressed pathogenic osteoclastogenesis in CD14+ monocytes from osteoporosis patients. These results identify PPP1R15A as a novel regulator of osteoclastogenesis and a valuable therapeutic target for osteoporosis.


Assuntos
Guanabenzo , Osteoporose , Animais , Feminino , Humanos , Camundongos , Diferenciação Celular , Guanabenzo/análogos & derivados , Guanabenzo/uso terapêutico , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos , Osteogênese , Osteoporose/tratamento farmacológico , Ovariectomia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/farmacologia , Ligante RANK/metabolismo
8.
Cell Commun Signal ; 22(1): 65, 2024 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-38267954

RESUMO

Protein Phosphatase 1 (PP1) is a major serine/threonine phosphatase in eukaryotes, participating in several cellular processes and metabolic pathways. Due to their low substrate specificity, PP1's catalytic subunits do not exist as free entities but instead bind to Regulatory Interactors of Protein Phosphatase One (RIPPO), which regulate PP1's substrate specificity and subcellular localization. Most RIPPOs bind to PP1 through combinations of short linear motifs (4-12 residues), forming highly specific PP1 holoenzymes. These PP1-binding motifs may, hence, represent attractive targets for the development of specific drugs that interfere with a subset of PP1 holoenzymes. Several viruses exploit the host cell protein (de)phosphorylation machinery to ensure efficient virus particle formation and propagation. While the role of many host cell kinases in viral life cycles has been extensively studied, the targeting of phosphatases by viral proteins has been studied in less detail. Here, we compile and review what is known concerning the role of PP1 in the context of viral infections and discuss how it may constitute a putative host-based target for the development of novel antiviral strategies.


Assuntos
Processamento de Proteína Pós-Traducional , Viroses , Humanos , Proteína Fosfatase 1 , Fosforilação , Fatores de Transcrição , Holoenzimas
9.
Clin Transl Oncol ; 26(1): 119-135, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37261660

RESUMO

BACKGROUND: Protein phosphatase 1 regulatory subunit 14B (PPP1R14B) is an oncogenic gene found in a variety of tumors, but its role in the prognosis and development of kidney renal clear cell carcinoma (KIRC) remains unknown. Our study aimed to determine whether PPP1R14B could be a prognostic biomarker for KIRC and its role in the development of KIRC. METHODS: In this work, we used The Cancer Genome Atlas (TCGA) database to explore the expression of PPP1R14B in tumor tissues, its relationship with the prognosis of tumor patients, and its role in tumor occurrence and development. We validated our findings using the International Cancer Genome Consortium (ICGC) cohort, our clinical samples, and in vitro experiments. RESULTS: PPP1R14B was upregulated in KIRC compared to adjacent normal tissue. Moreover, multivariate analysis revealed that upregulated PPP1R14B expression was an independent risk factor for KIRC progression. High-PPP1R14B groups had shorter overall survival (OS) and disease-free survival (DFS) in TCGA and ICGC cohorts. We used Cell Counting Kit-8 (CCK8) and scratch wound healing assay to explore the proliferation and migration of KIRC cells following PPP1R14B knockdown. Our results indicated that PPP1R14B knockdown significantly reduced the proliferation and migration of KIRC cells in vitro. We also explored the possible cellular mechanisms of PPP1R14B through the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene ontology (GO) analysis, and TISIDB analysis. The function enrich analysis revealed that PPP1R14B-related genes were mainly enriched in purine metabolism and the macromolecule catabolic process. PPP1R14B expression was associated with tumor-infiltrating immune cells (TIICs) in the TCGA cohort, and the results of single-cell RNA-seq (scRNA) further demonstrated that PPP1R14B expression was associated with the enhanced infiltration of CD8 + T lymphocytes. CONCLUSION: PPP1R14B may serve as a prognostic biomarker in KIRC, affect purine metabolism, activate immune infiltration, and promote KIRC cell migration.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Biomarcadores , Carcinoma de Células Renais/genética , Rim , Neoplasias Renais/genética , Prognóstico , Proteína Fosfatase 1 , Purinas
10.
J Biol Chem ; 300(1): 105515, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38042495

RESUMO

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is ∼10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1's M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.


Assuntos
Domínio Catalítico , Proteína Fosfatase 1 , Ubiquitina-Proteína Ligases , Ligação Proteica , Proteína Fosfatase 1/química , Humanos , Ubiquitina-Proteína Ligases/química , Microscopia Crioeletrônica , Metais/química
11.
Am J Physiol Renal Physiol ; 326(2): F285-F299, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38096266

RESUMO

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.


Assuntos
Arginina Vasopressina , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Células HEK293 , Arginina Vasopressina/metabolismo , Desamino Arginina Vasopressina , Colforsina , Proteína Fosfatase 1/metabolismo , Rim/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo
12.
Mol Cell ; 84(3): 506-521.e11, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38159565

RESUMO

Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.


Assuntos
Domínio Catalítico , Fator de Iniciação 2 em Eucariotos , Proteína Fosfatase 1 , Humanos , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
13.
J Exp Clin Cancer Res ; 42(1): 334, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38057879

RESUMO

BACKGROUND: With the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored. METHODS: Differential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A. RESULTS: Our analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation. CONCLUSIONS: Our study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.


Assuntos
Neoplasias Colorretais , MicroRNAs , Humanos , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Hibridização in Situ Fluorescente , MicroRNAs/genética , Proteína Fosfatase 1/metabolismo , RNA/genética , RNA Circular/genética , RNA Circular/metabolismo
14.
Biomolecules ; 13(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-38136612

RESUMO

Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin-proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1's inhibitory potency. Inhibiting PP1 to mimic PHI-1's function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.


Assuntos
Sistema de Sinalização das MAP Quinases , Neoplasias , Humanos , Proteína Fosfatase 1 , Sistema de Sinalização das MAP Quinases/fisiologia , Proteostase , Células HEK293 , Quinases de Proteína Quinase Ativadas por Mitógeno
15.
Int J Mol Sci ; 24(24)2023 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-38139150

RESUMO

The vertebrate PPP1R15 family consists of the proteins GADD34 (growth arrest and DNA damage-inducible protein 34, the product of the PPP1R15A gene) and CReP (constitutive repressor of eIF2α phosphorylation, the product of the PPP1R15B gene), both of which function as targeting/regulatory subunits for protein phosphatase 1 (PP1) by regulating subcellular localization, modulating substrate specificity and assembling complexes with target proteins. The primary cellular function of these proteins is to facilitate the dephosphorylation of eukaryotic initiation factor 2-alpha (eIF2α) by PP1 during cell stress. In this review, we will provide a comprehensive overview of the cellular function, biochemistry and pharmacology of GADD34 and CReP, starting with a brief introduction of eIF2α phosphorylation via the integrated protein response (ISR). We discuss the roles GADD34 and CReP play as feedback inhibitors of the unfolded protein response (UPR) and highlight the critical function they serve as inhibitors of the PERK-dependent branch, which is particularly important since it can mediate cell survival or cell death, depending on how long the stressful stimuli lasts, and GADD34 and CReP play key roles in fine-tuning this cellular decision. We briefly discuss the roles of GADD34 and CReP homologs in model systems and then focus on what we have learned about their function from knockout mice and human patients, followed by a brief review of several diseases in which GADD34 and CReP have been implicated, including cancer, diabetes and especially neurodegenerative disease. Because of the potential importance of GADD34 and CReP in aspects of human health and disease, we will discuss several pharmacological inhibitors of GADD34 and/or CReP that show promise as treatments and the controversies as to their mechanism of action. This review will finish with a discussion of the biochemical properties of GADD34 and CReP, their regulation and the additional interacting partners that may provide insight into the roles these proteins may play in other cellular pathways. We will conclude with a brief outline of critical areas for future study.


Assuntos
Doenças Neurodegenerativas , Proteína Fosfatase 1 , Animais , Humanos , Camundongos , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos Knockout , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismo
16.
Int J Mol Sci ; 24(24)2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-38139189

RESUMO

TIMAP (TGF-ß-inhibited membrane associated protein) is abundant in endothelial cells, and it has been regarded as a member of the myosin phosphatase targeting protein (MYPT) family. Our workgroup previously identified several interacting protein partners of TIMAP and proved its regulatory subunit role for protein phosphatase 1 catalytic subunit (PP1c). TIMAP is also expressed in neuronal cells, but details of its function have not been studied yet. Therefore, we aimed to explore the role of TIMAP in neuronal cells, especially during differentiation. Expression of TIMAP was proved both at mRNA and protein levels in SH-SY5Y human neuroblastoma cells. Differentiation of SH-SY5Y cells was optimized and proved by the detection of neuronal differentiation markers, such as ß3-tubulin, nestin and inhibitor of differentiation 1 (ID1) using qPCR and Western blot. We found downregulation of TIMAP during differentiation. In accordance with this, overexpression of recombinant TIMAP attenuated the differentiation of neuronal cells. Moreover, the subcellular localization of TIMAP has changed during differentiation as it translocated from the plasma membrane into the nucleus. The nuclear interactome of TIMAP revealed more than 50 proteins, offering the possibility to further investigate the role of TIMAP in several key physiological pathways of neuronal cells.


Assuntos
Células Endoteliais , Neurônios , Proteína Fosfatase 1 , Humanos , Diferenciação Celular , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Neurônios/citologia
17.
EMBO Rep ; 24(12): e56997, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37975164

RESUMO

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.


Assuntos
Proteínas de Drosophila , Proteínas de Membrana , Animais , Polaridade Celular/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
18.
J Biol Chem ; 299(12): 105432, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37926279

RESUMO

Phosphoprotein phosphatase 1 (PP1) associates with specific regulatory subunits to achieve, among other functions, substrate selectivity. Among the eight PP1 isotypes in Leishmania, PP1-8e associates with the regulatory protein PNUTS along with the structural factors JBP3 and Wdr82 in the PJW/PP1 complex that modulates RNA polymerase II (pol II) phosphorylation and transcription termination. Little is known regarding interactions involved in PJW/PP1 complex formation, including how PP1-8e is the selective isotype associated with PNUTS. Here, we show that PNUTS uses an established RVxF-ΦΦ-F motif to bind the PP1 catalytic domain with similar interfacial interactions as mammalian PP1-PNUTS and noncanonical motifs. These atypical interactions involve residues within the PP1-8e catalytic domain and N and C terminus for isoform-specific regulator binding. This work advances our understanding of PP1 isoform selectivity and reveals key roles of PP1 residues in regulator binding. We also explore the role of PNUTS as a scaffold protein for the complex by identifying the C-terminal region involved in binding JBP3 and Wdr82 and impact of PNUTS on the stability of complex components and function in pol II transcription in vivo. Taken together, these studies provide a potential mechanism where multiple motifs within PNUTS are used combinatorially to tune binding affinity to PP1, and the C terminus for JBP3 and Wdr82 association, in the Leishmania PJW/PP1 complex. Overall, our data provide insights in the formation of the PJW/PP1 complex involved in regulating pol II transcription in divergent protozoans where little is understood.


Assuntos
Proteínas de Ligação a DNA , Leishmania , Proteínas Nucleares , Proteína Fosfatase 1 , Animais , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Leishmania/genética , Leishmania/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
19.
Aging (Albany NY) ; 15(20): 11033-11051, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37837551

RESUMO

Growing evidence underscores the circadian rhythm's essential function in liver stability and disease. Its disruption is progressively linked with metabolic issues, oncogene triggers, and heightened cancer susceptibility. Research points to slingshot protein phosphatase 1 (SSH1), a modulator of cofilin-1 (CFL-1), as instrumental in the reformation of the actin cytoskeleton, thereby impacting the invasiveness of various cancer types. Yet, the dynamics of SSH1's influence on liver cell stemness and circadian activity remain unclear. Through in-silico, tissue analysis, and functional assays, the study reveals a significant SSH1 expression in HCC samples, compared to non-cancerous counterparts, across six HCC platforms (AUC between 0.62 and 0.77, p < 0.01). The aberrant expression of SSH1 was correlated with poor patients' survival (HR = 1.70, p = 0.0063) and progression-free (HR = 1.477, p = 0.0187) survival rates. Targeting SSH1, either via Sennoside A or CRISPR SSH1 in Huh7 cells (Huh7-SSH1-/-) significantly suppressed cell viability, migration, invasion, colony and tumorsphere formation of the Huh7-SSH1-/- cells. Mechanistically, we showed that downregulated SSH1 expression suppressed CLOCK, BMAL1, WNT3, ß-catenin, LRP5/6, BCL2, VIM and Snail, with concomitant upregulated CFL-1/2, and CRY1 expression, indicating dysregulated circadian rhythm and WNT/ß-catenin oncogenic pathway deactivation. Treatments in reflected notable tumor size reductions in the mice treated with SenAlight (1.76-fold, p < 0.01) and SenAdark (3.79-fold, p < 0.01). The expression of SSH1, CLOCK, BMAL1 and ß-catenin proteins were significantly downregulated in the SenAlight and SenAdark mice; this was more so in the SenAdark mice. This reveals a potential treatment approach for HCC patients.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/genética , Proteína Fosfatase 1 , beta Catenina , Via de Sinalização Wnt , Fatores de Transcrição ARNTL , Neoplasias Hepáticas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Fosfoproteínas Fosfatases
20.
Cells ; 12(20)2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37887268

RESUMO

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose , Camundongos , Animais , Proteína Fosfatase 1/metabolismo , Domínio Catalítico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Isoformas de Proteínas/metabolismo , Colágeno , Fibrinogênio/metabolismo
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