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1.
Medicine (Baltimore) ; 99(33): e21706, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32872046

RESUMO

MicroRNAs (miRNAs) have been suggested to act critical roles in the pathophysiology of traumatic osteonecrosis of the femoral head (TONFH). Unfortunately, their roles in the development of TONFH are still ambiguous. The purpose of this study is to identify promising miRNA biomarkers in traumatic osteonecrosis development.We conducted a comprehensive bioinformatics analysis using microarray datasets downloaded from the Gene Expression Omnibus database, and compared the expression of miRNAs in the serum of TONFH patients with controls. Next, we performed target prediction, function enrichment analysis, and protein-protein interaction network analysis based on differentially expressed (DE) miRNAs.We identified 26 DE miRNAs that may contribute to the pathophysiology of TONFH. The miRNAs were linked to ubiquitin proteasome system including conjugating protein ligase activity, ubiquitin-protein ligase activity and ubiquitin mediated proteolysis 5 pathway, and we exposed miR-181a-5p and miR-140-5p as promising biomarkers in TONFH.A predicting model consisting of 5 miRNAs may help discriminating high-risk patients who might develop TONFH after femur neck fracture. Among DE miRNAs, MiR-181a-5p and miR-140-5p may contribute to the development femoral head osteonecrosis after femur neck fracture via ubiquitin proteasome system.


Assuntos
Fraturas do Colo Femoral/genética , Necrose da Cabeça do Fêmur/genética , MicroRNAs/análise , Ubiquitina/genética , Biomarcadores/metabolismo , Biologia Computacional , Feminino , Fraturas do Colo Femoral/cirurgia , Perfilação da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética
2.
PLoS Genet ; 16(7): e1008933, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692737

RESUMO

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Fase S/genética , Schizosaccharomyces/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
3.
PLoS Pathog ; 16(6): e1008640, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569299

RESUMO

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.


Assuntos
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Ubiquitina/genética
5.
Nucleic Acids Res ; 48(11): 6310-6325, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32356875

RESUMO

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Mutação , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Poliubiquitina/química , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Especificidade por Substrato , Sumoilação , Ubiquitina/química , Ubiquitina/genética
6.
PLoS One ; 15(5): e0227786, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433703

RESUMO

Homeostasis is an essential concept to understand the stability of organisms and their adaptive behaviors when coping with external and internal assaults. Many hormones that take part in homeostatic control come in antagonistic pairs, such as glucagon and insulin reflecting the inflow and outflow compensatory mechanisms to control a certain internal variable, such as blood sugar levels. By including negative feedback loops homeostatic controllers can exhibit oscillations with characteristic frequencies. In this paper we demonstrate the associated frequency changes in homeostatic systems when individual controllers -in a set of interlocked feedback loops- gain control in response to environmental changes. Taking p53 as an example, we show how Per2, ATM and Mdm2 feedback loops -interlocked with p53- gain individual control in dependence to the level of DNA damage, and how each of these controllers provide certain functionalities in their regulation of p53. In unstressed cells, the circadian regulator Per2 ensures a basic p53 level to allow its rapid up-regulation in case of DNA damage. When DNA damage occurs the ATM controller increases the level of p53 and defends it towards uncontrolled degradation, which despite DNA damage, would drive p53 to lower values and p53 dysfunction. Mdm2 on its side keeps p53 at a high but sub-apoptotic level to avoid premature apoptosis. However, with on-going DNA damage the Mdm2 set-point is increased by HSP90 and other p53 stabilizers leading finally to apoptosis. An emergent aspect of p53 upregulation during cell stress is the coordinated inhibition of ubiquitin-independent and ubiquitin-dependent degradation reactions. Whether oscillations serve a function or are merely a by-product of the controllers are discussed in view of the finding that homeostatic control of p53, as indicated above, does in principle not require oscillatory homeostats.


Assuntos
Retroalimentação Fisiológica , Homeostase/genética , Modelos Teóricos , Proteína Supressora de Tumor p53/genética , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ritmo Circadiano/genética , Dano ao DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Proteínas Circadianas Period/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Ubiquitina/genética
7.
Mem Inst Oswaldo Cruz ; 115: e190242, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130365

RESUMO

BACKGROUND: Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE: To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS: We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS: We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the ß-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS: Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.


Assuntos
Giardia lamblia/metabolismo , Ubiquitina/genética , Ubiquitinação , Ubiquitinas/genética , Giardia lamblia/genética , Modelos Moleculares , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinas/metabolismo
8.
Nat Struct Mol Biol ; 27(4): 323-332, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203490

RESUMO

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
9.
Proc Natl Acad Sci U S A ; 117(14): 7776-7781, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193351

RESUMO

The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.


Assuntos
Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , Proteínas de Transporte Vesicular/genética , Poliubiquitina/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Homologia de Sequência
10.
Nat Commun ; 11(1): 1306, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161257

RESUMO

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.


Assuntos
Lamina Tipo A/metabolismo , Pseudogenes/genética , Ubiquitina/genética , Sistemas CRISPR-Cas/genética , Divisão Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Conjuntos de Dados como Assunto , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteômica , RNA-Seq , Ubiquitina/metabolismo , Ubiquitinação
11.
Nucleic Acids Res ; 48(6): 3042-3052, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32009145

RESUMO

Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.


Assuntos
Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ubiquitinação/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Poliubiquitina/genética , Mapas de Interação de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética
12.
Nat Struct Mol Biol ; 27(3): 240-248, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066963

RESUMO

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.


Assuntos
Reparo do DNA , DNA/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ubiquitina/química , Animais , Sítios de Ligação , Galinhas , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
13.
Cell Physiol Biochem ; 54(1): 1-14, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31916733

RESUMO

BACKGROUND/AIMS: Deubiquitinating enzymes (DUBs) are crucially involved in controlling signal transductions, and reverse ubiquitination by removing the ubiquitin from protein substrates. The Hippo signaling has an important role in tissue growth, cell proliferation, differentiation, and apoptosis. Since disruption of the Hippo signaling is associated with a number of diseases, it is imperative to investigate the molecular mechanism of the Hippo signaling. METHODS: DUB screening was performed using the kidney of the mouse unilateral ureteric obstruction (UUO) model to identify the cellular mechanism of the DUB-regulated Hippo signaling. In addition, kidney cells were used to confirm cell proliferation and protein levels in the Hippo signaling pathway. Densitometric analysis was conducted to compare the expression level of proteins using Image J. RESULTS: We found that YOD1, also known as OTU1, is downregulated in the mouse UUO model. We also demonstrated that YOD1 binds to and deubiquitinates neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4). Furthermore, we observed that YOD1 suppresses NEDD4-induced cell proliferation. CONCLUSION: YOD1 regulates the Hippo signaling pathway through NEDD4, and the K63-linked polyubiquitin chain of NEDD4 plays an important role. Also, our results indicate that YOD1 plays an important role in kidney diseases.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Mutagênese , Ubiquitina-Proteína Ligases Nedd4/química , Ubiquitina-Proteína Ligases Nedd4/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Tioléster Hidrolases/química , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
14.
PLoS One ; 15(1): e0227841, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945107

RESUMO

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.


Assuntos
Peptídeos/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Alanina/genética , DNA Polimerase III/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Treonina/genética , Ubiquitina/genética
15.
Nat Commun ; 11(1): 477, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980598

RESUMO

Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure. Three proteasome subunits-Rpn10, Rpn13, and Rpn1-can recognize ubiquitin chains. Here we report that proteins with single chains of K48-linked ubiquitin are targeted for degradation almost exclusively through binding to Rpn10. Rpn1 can act as a co-receptor with Rpn10 for K63 chains and for certain other chain types. Differences in targeting do not correlate with chain affinity to receptors. Surprisingly, in steady-state assays Rpn13 retarded degradation of various single-chain substrates. Substrates with multiple short ubiquitin chains can be presented for degradation by any of the known receptors, whereas those targeted to the proteasome through a ubiquitin-like domain are degraded most efficiently when bound by Rpn13 or Rpn1. Thus, the proteasome provides an unexpectedly versatile binding platform that can recognize substrates targeted for degradation by ubiquitin chains differing greatly in length and topology.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Sítios de Ligação , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas , Proteólise , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Ubiquitina/genética
16.
Gene ; 726: 144186, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31647998

RESUMO

Parkinson's disease (PD) is characterized by progressive death of dopamine producing neurons in the substantia nigra pars compacta of the mid brain. Dysfunction of an E3 ligase protein, Parkin, encoded by PARK2 gene, results in accumulation of misfolded proteins in brain cells which lead to the onset of PD. Parkin is a multi-domain protein consisting of N-terminal ubiquitin-like domain (Ubl) followed by RING0, RING1, In Between Ring (IBR) domain and RING2 domain which is present at the C-terminal end of Parkin protein. Ubl domain is the smallest domain of Parkin and is involved in the binding of Parkin with E2 protein molecule required for proper Ubiquitination and functioning of proteins in the brain. Mutations in the Parkin protein are known to be associated with protein dysfunction leading to PD. This study aims to decipher the characteristics and effects of the different mutations in the Ubl domain by an in-silico analysis. The mutations were collected from PDmutDB and COSMIC databases. The pathogenic impacts of amino-acid mutations on Ubl structure and function were analysed by using various computational tools. Due to lack of proper full-chain structure of the Ubl domain, a homology model of the domain was reconstructed using Discovery Studio 2.5 (DS 2.5) software suite. We found that the mutations A31D, A46P, C59F, A46T, E28K, E49K, R42P, R42S, and Q63K were the most deleterious ones which might be associated with the onset of PD. In order to study the dynamic behaviour of the Parkin Ubl domain in cellular environment, molecular dynamics (MD) simulations were carried out using the wild-type and mutant Ubl domains. Our analyses could predict the cellular dynamics of the mutations and therefore might help in predicting the hitherto unknown molecular mechanism of the disease onset and designing precision medicine for the treatment of PD.


Assuntos
Mutação/genética , Doença de Parkinson/genética , Domínios Proteicos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina/genética , Aminoácidos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína/genética , Ubiquitinação/genética
17.
Nucleic Acids Res ; 48(1): 231-248, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31722399

RESUMO

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição Genética , Ubiquitina/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Raios Ultravioleta
18.
J Neuropathol Exp Neurol ; 79(1): 34-45, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31750913

RESUMO

Guam parkinsonism-dementia (G-PD) is a progressive and fatal neurodegenerative disorder among the native inhabitants of the Mariana Islands that manifests clinically with parkinsonism as well as dementia. Neuropathologically, G-PD is characterized by abundant neurofibrillary tangles composed of hyperphosphorylated tau, marked deposition of transactive response DNA-binding protein 43 kDa (TDP-43), and neuronal loss. The mechanisms that underlie neurodegeneration in G-PD are poorly understood. Here, we report that the unfolded protein response (UPR) is activated in G-PD brains. Specifically, we show that the endoplasmic reticulum (ER) chaperone binding immunoglobulin protein/glucose-regulated protein 78 kDa and phosphorylated (activated) ER stress sensor protein kinase RNA-like ER kinase accumulate in G-PD brains. Furthermore, proteinaceous aggregates in G-PD brains are found to contain several proteins related to the ubiquitin-proteasome system (UPS) and the autophagy pathway, two major mechanisms for intracellular protein degradation. In particular, a mutant ubiquitin (UBB+1), whose presence is a marker for UPS dysfunction, is shown to accumulate in G-PD brains. We demonstrate that UBB+1 is a potent modifier of TDP-43 aggregation and cytotoxicity in vitro. Overall, these data suggest that UPR activation and intracellular proteolytic pathways are intimately connected with the accumulation of aggregated proteins in G-PD.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Deficiências na Proteostase/patologia , Resposta a Proteínas não Dobradas , Idoso , Idoso de 80 Anos ou mais , Esclerose Amiotrófica Lateral/genética , Autofagia , Encéfalo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Deficiências na Proteostase/genética , Transdução de Sinais/genética , Ubiquitina/genética , Ubiquitina/metabolismo
19.
Cancer Lett ; 469: 301-309, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31705931

RESUMO

Small ubiquitin-like modifier (SUMO), a critical regulatory modification protein, is involved in various biological processes, such as gene expression, genome maintenance and DNA damage repair (DDR). Numerous recent studies have revealed that disturbed SUMOylation and deSUMOylation homeostasis contribute to tumorigenesis. Abnormal alterations of key factors in the SUMO modification are closely related to cancer development and progression, indicating that the restoration of SUMOylation homeostasis may serve as a promising therapeutic strategy. In this review, we summarize the process and function of SUMOylation, clarify the 'dual-edged sword' functions of SUMOylation in diversified cancers and put forth future research prospects.


Assuntos
Carcinogênese/genética , Neoplasias/genética , Sumoilação/genética , Reparo do DNA/genética , Homeostase/genética , Humanos , Neoplasias/patologia , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genética , Ubiquitina/genética
20.
PLoS Genet ; 15(11): e1008387, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738769

RESUMO

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Microautofagia/genética , Complexo de Endopeptidases do Proteassoma/genética , Citoplasma/genética , Citoplasma/metabolismo , Glucose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Inanição/genética , Inanição/metabolismo , Ubiquitina/genética , Ubiquitinação/genética , Vacúolos/genética , Vacúolos/metabolismo
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