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1.
Adv Exp Med Biol ; 1164: 63-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576540

RESUMO

Gankyrin (also called PSMD10, p28, or p28GANK) is a crucial oncoprotein that is upregulated in various cancers and assumed to play pivotal roles in the initiation and progression of tumors. Although the in vitro function of gankyrin is relatively well characterized, its role in vivo remains to be elucidated. We have investigated the function of gankyrin in vivo by producing mice with liver parenchymal cell-specific gankyrin ablation (Alb-Cre;gankyrinf/f) and gankyrin deletion both in liver parenchymal and in non-parenchymal cells (Mx1-Cre;gankyrinf/f). Gankyrin deficiency both in non-parenchymal cells and parenchymal cells, but not in parenchymal cells alone, reduced STAT3 activity, interleukin-6 production, and cancer stem cell marker expression, leading to attenuated tumorigenic potential in the diethylnitrosamine hepatocarcinogenesis model. Essentially similar results were obtained by analyzing mice with intestinal epithelial cell-specific gankyrin ablation (Villin-Cre;Gankyrinf/f) and gankyrin deletion both in myeloid and epithelial cells (Mx1-Cre;Gankyrinf/f) in the colitis-associated cancer model. Clinically, gankyrin expression in the tumor microenvironment was negatively correlated with progression-free survival in patients undergoing treatment with Sorafenib for hepatocellular carcinomas. These findings indicate important roles played by gankyrin in non-parenchymal cells as well as parenchymal cells in the pathogenesis of liver cancers and colorectal cancers, and suggest that by acting both on cancer cells and on the tumor microenvironment, anti-gankyrin agents would be promising as therapeutic and preventive strategies against various cancers, and that an in vitro cell culture models that incorporate the effects of non-parenchymal cells and gankyrin would be useful for the study of human cell transformation.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Complexo de Endopeptidases do Proteassoma , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatologia , Carcinoma Hepatocelular/terapia , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Deleção de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Neoplasias Hepáticas/terapia , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral
2.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
3.
Plant Cell Physiol ; 60(8): 1633-1645, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292642

RESUMO

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Giberelinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Temperatura Ambiente , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Mem Inst Oswaldo Cruz ; 114: e190052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166481

RESUMO

BACKGROUND: Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE: The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS: The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS: 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS: Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.


Assuntos
Biomphalaria/genética , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , Animais , Biomphalaria/enzimologia , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Filogenia , Valores de Referência , Transcriptoma , Ubiquitinação
5.
Int J Mol Sci ; 20(9)2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075988

RESUMO

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.


Assuntos
Mutação/genética , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Humanos , Proteínas Mutantes/química , Multimerização Proteica
6.
Mol Med Rep ; 19(6): 4561-4568, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942447

RESUMO

Osteoarthritis (OA) is a common inflammatory joint disease. MicroRNAs (miRNAs/miRs) have been reported to be involved in the pathogenesis of OA; however, the role of miRNAs in OA remains largely unexplained. The purpose of the present study was to investigate the expression and role of miR­195­5p in OA, and to further explore the mechanism. The expression level of miR­195­5p was measured using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). TargetScan and a luciferase reporter assay were used to reveal the associations between miR­195­5p and REGγ (also known as PSME3). To investigate the role of miR­195­5p in OA, a cell model of OA was established by treating ATDC5 cells with lipopolysaccharide (LPS). Then an MTT assay was conducted to detect cell proliferation ability, and an Annexin V­fluorescein isothiocyanate/propidium iodide apoptosis detection kit was used to measure cell apoptosis. In addition, the levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α were determined using ELISA. Furthermore, gene and protein expression was measured via RT­qPCR and western blot assay, respectively. The results revealed that miR­195­5p was significantly upregulated in the articular cartilage tissues of patients with OA and in LPS stimulated ATDC5 cells. REGγ was a direct target of miR­195­5p. The repressed cell proliferation ability and enhanced cell apoptosis of ATDC5 cells induced by LPS were reversed by miR­195­5p downregulation. Furthermore, LPS stimulation significantly upregulated the levels of IL­1ß, IL­6 and TNF­α, while miR­195­5p downregulation markedly reduced the expression of inflammatory factors induced by LPS. The results also revealed that a miR­195­5p inhibitor inhibited the LPS induced repression of the Wnt/ß­catenin signaling pathway and activation of nuclear factor (NF)­κB signaling pathway in ATDC5 cells. Notably, the results of the present study also indicated that all of the effects of the miR­195­5p inhibitor on ATDC5 cells were reversed by REGγ silencing. In conclusion, the results indicated that the miR­195­5p inhibitor served a protective role in OA by inhibiting chondrocyte apoptosis and inflammatory responses by regulating the Wnt/ß­catenin and NF­κB signaling pathways.


Assuntos
Autoantígenos/genética , MicroRNAs/genética , Osteoartrite/terapia , Complexo de Endopeptidases do Proteassoma/genética , Adulto , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoartrite/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Via de Sinalização Wnt
7.
Cell Mol Life Sci ; 76(14): 2761-2777, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31030225

RESUMO

Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems suffer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Targeted proteolysis has the potential to overcome some of these limitations. The field of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specific proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the different targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics.


Assuntos
Edição de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Ubiquitinação
8.
J Exp Clin Cancer Res ; 38(1): 176, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023317

RESUMO

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is the most lethal of all gynecological malignancies. Patients often suffer from chemoresistance. Several studies have reported that Fn14 could regulate migration, invasion, and angiogenesis in cancer cells. However, its functional role in chemoresistance of HGSOC is still unknown. METHODS: The expression of Fn14 in tissue specimens was detected by IHC. CCK-8 assay was performed to determine changes in cell viability. Apoptosis was measured by flow cytometry. The potential molecular mechanism of Fn14-regulated cisplatin resistance in HGSOC was investigated using qRT-PCR, western blotting, and Co-IP assays. The role of Fn14 in HGSOC was also investigated in a xenograft mouse model. RESULTS: In this study, we found that Fn14 was significantly downregulated in patients with cisplatin resistance. Patients with low Fn14 expression were associated with shorter progression-free survival and overall survival. Overexpression of Fn14 suppressed cisplatin resistance in OVCAR-3 cells, whereas knockdown of Fn14 did not affect cisplatin resistance in SKOV-3 cells. Interestingly, Fn14 could exert anti-chemoresistance effect only in OVCAR-3 cells harboring a p53-R248Q mutation, but not in SKOV-3 cells with a p53-null mutation. We isolated and identified primary cells from two patients with the p53-R248Q mutation from HGSOC patients and the anti-chemoresistance effect of Fn14 was observed in both primary cells. Mechanistic studies demonstrated that overexpression of Fn14 could reduce the formation of a Mdm2-p53-R248Q-Hsp90 complex by downregulating Hsp90 expression, indicating that degradation of p53-R248Q was accelerated via Mdm2-mediated ubiquitin-proteasomal pathway. CONCLUSION: Our findings demonstrate for the first time that Fn14 overcomes cisplatin resistance through modulation of the degradation of p53-R248Q and restoration of Fn14 expression might be a novel strategy for the treatment of HGSOC.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/genética , Receptor de TWEAK/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Intervalo Livre de Progressão , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Gene ; 702: 66-74, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30930224

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with high mortality and frequent recurrence. Although various therapies provide potential cure for HCC patients, unfortunately the five-year survival rate of advanced HCC remains dismal. It is critical to explore the pathogenesis of HCC and identify novel biomarkers for early HCC diagnosis. PSMD4 is a major receptor of the 26S proteasome involved in ubiquitindependent and proteasome-mediated protein degradation. In our study, PSMD4 was overexpressed in HCC tissues and cell lines determined by Northern blot, western blot and immunohistochemistry. The silencing of PSMD4 blocked cell proliferation and tumor growth, induced cell apoptosis and inhibited the proteasome activity. Western blot results showed that the knockdown of PSMD4 blocked the expression of cyclooxygenase 2 (COX2), phosphorylated Sarcoma tyrosine kinase (P-SRC) and Bcl-2, but improved the levels of p53 and Bax in HCC, lung cancer, colorectal cancer, breast cancer and endometrial cancer cell lines. Taken together, these findings indicated that the subunit of 26S proteasome PSMD4 exerts as an oncogene in HCC and other cancers via regulating the expression p53, Bcl-2 and Bax. These findings enriched the pathogenesis of HCC, and provided a new biomarker for cancers diagnosis and a new target for cancers therapy.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Animais , Apoptose , Carcinogênese , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA
10.
BMC Cancer ; 19(1): 253, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898113

RESUMO

BACKGROUND: Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. METHODS: We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. RESULTS: The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. CONCLUSIONS: Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.


Assuntos
Carcinoma Ductal Pancreático/genética , Oncogenes/genética , Neoplasias Pancreáticas/genética , Complexo de Endopeptidases do Proteassoma/genética , Bortezomib/farmacologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Humano/genética , Genômica/métodos , Humanos , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Inibidores de Proteassoma/farmacologia , RNA Interferente Pequeno/genética , Esferoides Celulares
11.
J Biol Chem ; 294(16): 6562-6577, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30814255

RESUMO

The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as 'Rpt ring', assembles through six ATPase subunits (Rpt1-Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2, and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early-stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism-degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully-formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone-mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation.


Assuntos
Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Plant Sci ; 280: 314-320, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824010

RESUMO

The 26S proteasome is a multi-subunit protease controlling most of the cytosolic and nuclear protein turnover, regulating many cellular events in eukaryotes. However, functional modification on this complex remains unclear. Here, we showed a novel mechanism that a SUMO ligase AtMMS21 regulates activity of the 26S proteasome in root development of Arabidopsis. Our in vitro and in vivo data supported that AtMMS21 interacts with RPT2a, a subunit of the 26S proteasome. The mutants of AtMMS21 and RPT2a display similar developmental defect of roots, suggesting their association in this process. In addition, RPT2a is modified by SUMO3, potentially related to AtMMS21. During development, the activity of the 26S proteasome is lower in both mutants of AtMMS21 and RPT2a, compared with that of wild type. Furthermore, the protein level but not the RNA level of RPT2a is decreased in the absence of AtMMS21, implying stability regulation of the proteasome complex through the AtMMS21-RPT2a interaction. Taken together, the current study would improve our understanding on the regulatory mechanism of the 26S proteasome via protein modification in root development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ligases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Ligases/genética , Mutação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/genética , Sumoilação
13.
Proc Natl Acad Sci U S A ; 116(8): 3202-3210, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30723150

RESUMO

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.


Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Proteínas de Choque Térmico/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Compostos de Amônio/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Nitrogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/patologia
14.
EBioMedicine ; 41: 320-332, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30745168

RESUMO

BACKGROUND: Hyper-activation of TGF-ß signaling is critically involved in progression of hepatocellular carcinoma (HCC). However, the events that contribute to the dysregulation of TGF-ß pathway in HCC, especially at the post-translational level, are not well understood. METHODS: Associations of deubiquitinase POH1 with TGF-ß signaling activity and the outcomes of HCC patients were examined by data mining of online HCC datasets, immunohistochemistry analyses using human HCC specimens, spearman correlation and survival analyses. The effects of POH1 on the ubiquitination and stability of the TGF-ß receptors (TGFBR1 and TGFBR2) and the activation of downstream effectors were tested by western blotting. Primary mouse liver tissues from polyinosinic:polycytidylic acid (poly I:C)- treated Mx-Cre+, poh1f/f mice and control mice were used to detect the TGF-ß receptors. The metastatic-related capabilities of HCC cells were studied in vitro and in mice. FINDINGS: Here we show that POH1 is a critical regulator of TGF-ß signaling and promotes tumor metastasis. Integrative analyses of HCC subgroups classified with unsupervised transcriptome clustering of the TGF-ß response, metastatic potential and outcomes, reveal that POH1 expression positively correlates with activities of TGF-ß signaling in tumors and with malignant disease progression. Functionally, POH1 intensifies TGF-ß signaling delivery and, as a consequence, promotes HCC cell metastatic properties both in vitro and in vivo. The expression of the TGF-ß receptors was severely downregulated in POH1-deficient mouse hepatocytes. Mechanistically, POH1 deubiquitinates the TGF-ß receptors and CAV1, therefore negatively regulates lysosome pathway-mediated turnover of TGF-ß receptors. CONCLUSION: Our study highlights the pathological significance of aberrantly expressed POH1 in TGF-ß signaling hyperactivation and aggressive progression in HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Caveolina 1/metabolismo , Neoplasias Hepáticas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Prognóstico , Complexo de Endopeptidases do Proteassoma/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/antagonistas & inibidores , Transativadores/genética , Ubiquitinação
15.
J Biol Chem ; 294(15): 5759-5773, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755480

RESUMO

Hepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased RRM2 mRNA and protein expression levels. Moreover, siRNA-mediated silencing of RRM2 suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. RRM2 knockdown reduced NS5B expression, which depended on the protein degradation pathway, as NS5B RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that RRM2 silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hepacivirus/fisiologia , Hepatite C Crônica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribonucleosídeo Difosfato Redutase/biossíntese , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Regulação Enzimológica da Expressão Gênica , Hepatite C Crônica/genética , Hepatite C Crônica/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Proteólise , Ribonucleosídeo Difosfato Redutase/genética , Ubiquitinação/genética , Proteínas não Estruturais Virais/genética
16.
Nat Commun ; 10(1): 926, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804369

RESUMO

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.


Assuntos
Infecções por HIV/metabolismo , Integrase de HIV/metabolismo , HIV-1/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Infecções por HIV/genética , Infecções por HIV/virologia , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Proteólise , Provírus/enzimologia , Provírus/genética , Provírus/fisiologia , Fatores de Transcrição/genética , Integração Viral
17.
PLoS Genet ; 15(2): e1007917, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30707697

RESUMO

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.


Assuntos
Proteínas de Ligação ao GTP/genética , Mamíferos/genética , Proteínas dos Microfilamentos/genética , Monossomia/genética , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Polirribossomos/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA/genética , RNA Mensageiro/genética , Ribossomos/genética , Serina-Treonina Quinases TOR/genética , Regulação para Cima/genética
18.
Mol Cancer ; 18(1): 25, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777076

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs with a loop structure, but its functions remain largely unknown. Growing evidence has revealed that circRNAs play a striking role as functional RNAs in the progression of malignant disease. However, the precise role of circRNAs in gastric cancer (GC) remains unclear. METHODS: CircRNAs were determined by human circRNA array analysis and quantitative reverse transcription polymerase reaction. Luciferase reporter, RNA pull down, and fluorescence in situ hybridization assays were employed to test the interaction between circPSMC3 and miR-296-5p. Ectopic over-expression and siRNA-mediated knockdown of circPSMC3, proliferation, migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPSMC3. RESULTS: CircPSMC3 rather than liner PSMC3 mRNA was down-regulated in GC tissues, corresponding plasmas from GC patients as well as GC cell lines compared to normal controls. Lower circPSMC3 expression in GC patients was correlated with higher TNM stage and shorter overall survival. Over-expression of circPSMC3 and miR-296-5p inhibitor could inhibit the tumorigenesis of gastric cancer cells in vivo and vitro whereas co-transfection of circPSMC3 and miRNA-296-5p could counteract this effect. Importantly, we demonstrated that circPSMC3 could act as a sponge of miR-296-5p to regulate the expression of Phosphatase and Tensin Homolog (PTEN), and further suppress the tumorigenesis of gastric cancer cells. CONCLUSION: Our study reveals that circPSMC3 can serve as a novel potential circulating biomarker for detection of GC. CircPSMC3 participates in progression of gastric cancer by sponging miRNA-296-5p with PTEN, providing a new insight into the treatment of gastric cancer.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA/genética , Neoplasias Gástricas/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Metástase Linfática , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA/antagonistas & inibidores , RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida
19.
FEMS Yeast Res ; 19(2)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629175

RESUMO

The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quinolonas/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Regulação para Cima , 4-Nitroquinolina-1-Óxido/metabolismo , Oxidantes/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Fisiológico
20.
Methods Mol Biol ; 1915: 149-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617802

RESUMO

As opposed to proteasome-mediated proteolysis that leads to protein degradation, calpain proteases carry out limited proteolytic cleavages of their substrates. The cleavage of some substrates can produce active fragments that perform functions that are different from those performed by the full-length proteins. Therefore, cleavage by calpains can operate as a posttranslational modification and increase the functional diversity of target proteins. Nevertheless, activation of protein function by calpain cleavage is still an understudied area in molecular biology. Identifying and functionally characterizing by products generated by calpain cleavage could lead to the discovery of biomarkers and the identification of novel drug targets for the treatment of human diseases. This chapter contains a workflow designed to experimentally characterize novel calpain substrates, including identification of potential calpain targets via Western blotting, characterization of calpain cleavage sites, and the study of cellular functions played by such cleaved products. We will employ MYC as an example for these experiments.


Assuntos
Calpaína/genética , Proteínas de Membrana/química , Biologia Molecular/métodos , Calpaína/química , Humanos , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/genética , Proteólise , Especificidade por Substrato
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