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1.
Nat Chem Biol ; 15(8): 786-794, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31320752

RESUMO

Protein-protein interactions between E3 ubiquitin ligases and protein termini help shape the proteome. These interactions are sensitive to proteolysis, which alters the ensemble of cellular N and C termini. Here we describe a mechanism wherein caspase activity reveals latent C termini that are then recognized by the E3 ubiquitin ligase CHIP. Using expanded knowledge of CHIP's binding specificity, we predicted hundreds of putative interactions arising from caspase activity. Subsequent validation experiments confirmed that CHIP binds the latent C termini at tauD421 and caspase-6D179. CHIP binding to tauD421, but not tauFL, promoted its ubiquitination, while binding to caspase-6D179 mediated ubiquitin-independent inhibition. Given that caspase activity generates tauD421 in Alzheimer's disease (AD), these results suggested a concise model for CHIP regulation of tau homeostasis. Indeed, we find that loss of CHIP expression in AD coincides with the accumulation of tauD421 and caspase-6D179. These results illustrate an unanticipated link between caspases and protein homeostasis.


Assuntos
Caspases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Caspases/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação
2.
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
3.
Microb Pathog ; 132: 362-368, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054366

RESUMO

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.


Assuntos
Flavivirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Patos , Flavivirus/efeitos dos fármacos , Flavivirus/patogenicidade , Técnicas de Silenciamento de Genes , Leupeptinas/antagonistas & inibidores , Doenças das Aves Domésticas/virologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Interferente Pequeno , Transfecção , Ubiquitina/efeitos dos fármacos , Ubiquitina/genética , Proteínas do Envelope Viral , Internalização do Vírus
4.
Nat Cell Biol ; 21(6): 731-742, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086261

RESUMO

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1ß release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.


Assuntos
Artrite/genética , Inflamação/genética , Fatores de Transcrição Kruppel-Like/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Artrite/induzido quimicamente , Artrite/patologia , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Mutação , NF-kappa B/genética , Necrose/genética , Necrose/patologia , Ligação Proteica , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Ubiquitina/genética
5.
Anim Sci J ; 90(6): 728-736, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31006927

RESUMO

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, ß-carotene, vitamin E and IGF-1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma ß-carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma ß-carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Bovinos/crescimento & desenvolvimento , Bovinos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Oryza , Proteólise , Silagem , Vitamina A/metabolismo , Vitamina E/metabolismo , beta Caroteno/metabolismo , Fenômenos Fisiológicos da Nutrição Animal/genética , Animais , Calpaína/genética , Calpaína/metabolismo , Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo
6.
In Vitro Cell Dev Biol Anim ; 55(5): 355-367, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30993557

RESUMO

N-terminal acetylation (Nt-acetylation) refers to the acetylation of the free α-amino group at the N-terminus of a polypeptide. While the effects of Nt-acetylation are multifaceted, its most known function is in the acetylation-dependent N-end rule protein degradation pathway (Ac/N-end rule pathway), where Nt-acetylation is recognized as a degron by designated E3 ligases, eventually leading to target degradation by the ubiquitin-proteasome system. Naa10 is the catalytic subunit of the major Nt-acetylation enzyme NatA, which Nt-acetylates proteins whose second amino acid has a small side chain. In humans, NAA10 is the responsible mutated gene in Ogden syndrome and is thought to play important roles in development. However, it is unclear how the Ac/N-end rule pathway affects the differentiation ability of mouse embryonic stem cells (mESCs). We hypothesized that the balance of pluripotency factors may be maintained by the Ac/N-end rule pathway. Thus, we established Naa10 knockout mESCs to test this hypothesis. We found that Naa10 deficiency attenuated differentiation towards the epiblast lineage, deviating towards primitive endoderm. However, this was not caused by disturbing the balance of pluripotency factors, rather by augmenting FGF/MAPK signaling.


Assuntos
Linhagem da Célula/genética , Camadas Germinativas/crescimento & desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Acetilação , Animais , Diferenciação Celular/genética , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Inativação de Genes , Camadas Germinativas/metabolismo , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteólise , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética
7.
Mol Cell ; 74(4): 742-757.e8, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30979586

RESUMO

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Doenças por Armazenamento dos Lisossomos/genética , Doenças Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteína com Valosina/genética , Adenosina Trifosfatases/genética , Animais , Autofagia/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Camundongos , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Fosforilação/genética , Estresse Fisiológico/genética , Ubiquitina/genética
8.
Mol Cell ; 74(2): 330-346.e11, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853400

RESUMO

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.


Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genética
9.
Mol Cell ; 74(2): 363-377.e5, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30879902

RESUMO

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.


Assuntos
Proteínas de Drosophila/genética , Longevidade/genética , Fator 6 Associado a Receptor de TNF/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Enzimas Desubiquitinantes , Drosophila/genética , Longevidade/fisiologia , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ubiquitina/genética
10.
BMC Res Notes ; 12(1): 188, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925931

RESUMO

OBJECTIVE: Prenylated Rab Acceptor 1 (PRA1) is a transmembrane protein localized to the early secretory pathway. It has been found to interact with an array of Rab GTPases, leading to its hypothesized function in the recycling of Rab GTPases. However, all previous strategies used to screen for novel interacting partners have utilized a classic yeast two-hybrid approach that requires both bait and its potential binding partners to be cytosolic proteins. In the split-ubiquitin yeast two-hybrid screen, a protein interaction leads to the re-constitution of ubiquitin, which is followed by proteolytic release of a transcription activator that migrates to the nucleus alone. This allows for bait and/or prey to be integral membrane protein(s). To better understand the in vivo function of PRA1, we took an unbiased approach that screened PRA1 against a normalized mouse neuronal cDNA library using this variant of the classic screening strategy. RESULTS: We report 41 previously unidentified potential PRA1 binding partners revealed by this screen and validate the screen by confirming three of these interactions using a bi-molecular fluorescence complementation assay in mammalian cells. The identified proteins reside throughout the secretory pathway and are both membrane-bound and cytosolic in their identity, suggesting alternative functions for PRA1.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Cercopithecus aethiops , Proteínas de Ligação ao GTP/genética , Biblioteca Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/metabolismo , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/genética
11.
Methods Enzymol ; 618: 49-72, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850062

RESUMO

Ubiquitin has seven lysines, all of which are used to generate polyubiquitin chains in the yeast Saccharomyces cerevisiae. While the biology associated with chains formed through lysines 48 and 63 is well studied, other chain types are more poorly characterized. We outline a methodology for using synthetic genetic analysis to examine ubiquitin mutants. Ubiquitin is encoded by four loci, necessitating several alterations to standard protocols, including the use of the SK1 strain background, which sporulates with very high efficiency. The methods described here could be used to examine other ubiquitin mutants, including those that do not support viability.


Assuntos
Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Lisina/genética , Lisina/metabolismo , Mutação , Poliubiquitina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , Ubiquitinação
12.
Mol Cell ; 74(3): 436-451.e7, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30926242

RESUMO

The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.


Assuntos
Inflamação/genética , Conformação Proteica , Ubiquitina Tiolesterase/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Inflamação/patologia , Mutação/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/genética , Transdução de Sinais/genética , Especificidade por Substrato , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Ubiquitina/genética , Ubiquitina Tiolesterase/química
13.
Mol Cell ; 74(3): 421-435.e10, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30926243

RESUMO

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.


Assuntos
Carcinogênese/genética , Neoplasias/genética , Ubiquitina Tiolesterase/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Mutação/genética , Neoplasias/tratamento farmacológico , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Ubiquitina/genética , Ubiquitina Tiolesterase/química
14.
Methods Enzymol ; 618: 1-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850047

RESUMO

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.


Assuntos
Histonas/síntese química , Ubiquitina/síntese química , Proteínas de Xenopus/síntese química , Xenopus laevis , Animais , Histonas/química , Histonas/genética , Hidrólise , Lisina/síntese química , Lisina/química , Lisina/genética , Modelos Moleculares , Ubiquitina/química , Ubiquitina/genética , Ubiquitinação , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genética
15.
J Exp Clin Cancer Res ; 38(1): 120, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850009

RESUMO

BACKGROUND: Cysteine-rich intestinal protein 1 (CRIP1) is highly expressed in human intestine and aberrantly expressed in several types of tumor. However, studies on CRIP1 are limited and its role on tumor development and progression remains controversial and elusive. METHODS: Immunohistochemistry was performed to evaluate the expression of CRIP1 in paired normal and colorectal tumor specimens, as well as colorectal cell lines. Functional assays, such as CCK8, TUNEL assay and in vivo tumor growth assay, were used to detect the proliferation, apoptosis and response to 5-FU of CRIP1. Western blot was used to analyze Fas-mediated pathway induced by CRIP1. Rescue experiments were performed to evaluate the essential role of CRIP1 for Fas-mediated apoptosis. RESULTS: We demonstrated that CRIP1 is overexpressed in CRC tissues compared with adjacent normal mucosa. CRIP1 could dramatically recover the 5-Fluorouracil (5-FU) inhibited CRC cell proliferation in vitro and stimulate the tumor formation of CRC in vivo, probably through inhibiting CRC cell apoptosis. Moreover, CRIP1 also dramatically recovered the 5-Fluorouracil (5-FU) induced tumor cell apoptosis in vitro. Further study demonstrated that CRIP1 down-regulated the expression of Fas protein and proteins related to Fas-mediated apoptosis. CRIP1 could interact with Fas protein and stimulate its ubiquitination and degradation. In addition, a negative correlation was detected between the expression of CRIP1 and Fas protein in most of the clinical human CRC samples. CONCLUSION: The current research reveals a vital role of CRIP1 in CRC progression, which provide a novel target for clinical drug resistance of colorectal cancer and undoubtedly contributing to the therapeutic strategies in CRC.


Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas com Domínio LIM/genética , Receptor fas/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/genética , Proteólise , Ubiquitina/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Phys Chem Chem Phys ; 21(20): 10217-10227, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-30860214

RESUMO

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.


Assuntos
Técnicas de Química Analítica/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Gadolínio/química , Proteínas/análise , Marcadores de Spin , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Mutação , Proteínas/química , Proteínas/genética , Ubiquitina/análise , Ubiquitina/genética
17.
Nucleic Acids Res ; 47(7): 3784-3794, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30753618

RESUMO

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Ubiquitina/química , Ubiquitinas/química , Fatores de Transcrição Winged-Helix/química , Sequência de Aminoácidos/genética , Sobrevivência Celular , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Conformação Proteica em alfa-Hélice/genética , Ubiquitina/genética , Ubiquitinas/genética , Raios Ultravioleta , Fatores de Transcrição Winged-Helix/genética
18.
Nat Genet ; 51(3): 387-393, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804566

RESUMO

Insomnia is a common disorder linked with adverse long-term medical and psychiatric outcomes. The underlying pathophysiological processes and causal relationships of insomnia with disease are poorly understood. Here we identified 57 loci for self-reported insomnia symptoms in the UK Biobank (n = 453,379) and confirmed their effects on self-reported insomnia symptoms in the HUNT Study (n = 14,923 cases and 47,610 controls), physician-diagnosed insomnia in the Partners Biobank (n = 2,217 cases and 14,240 controls), and accelerometer-derived measures of sleep efficiency and sleep duration in the UK Biobank (n = 83,726). Our results suggest enrichment of genes involved in ubiquitin-mediated proteolysis and of genes expressed in multiple brain regions, skeletal muscle, and adrenal glands. Evidence of shared genetic factors was found between frequent insomnia symptoms and restless legs syndrome, aging, and cardiometabolic, behavioral, psychiatric, and reproductive traits. Evidence was found for a possible causal link between insomnia symptoms and coronary artery disease, depressive symptoms, and subjective well-being.


Assuntos
Predisposição Genética para Doença/genética , Distúrbios do Início e da Manutenção do Sono/genética , Sono/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteólise , Autorrelato , Ubiquitina/genética
19.
PLoS One ; 14(2): e0209592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30789917

RESUMO

Initiation of treatment during the pre-symptomatic phase of Yersinia pestis (Y. pestis) infection is particularly critical. The rapid proliferation of Y. pestis typically couples with the manifestation of common flu-like early symptoms that often misguides the medical intervention. Our study used African green monkeys (AGM) that did not exhibit clear clinical symptoms for nearly two days after intranasal challenge with Y. pestis and succumbed within a day after showing the first signs of clinical symptoms. The lung, and mediastinal and submandibular lymph nodes (LN) accumulated significant Y. pestis colonization immediately after the intranasal challenge. Hence, organ-specific molecular investigations are deemed to be the key to elucidating mechanisms of the initial host response. Our previous study focused on the whole blood of AGM, and we found early perturbations in the ubiquitin-microtubule-mediated host defense. Altered expression of the genes present in ubiquitin and microtubule networks indicated an early suppression of these networks in the submandibular lymph nodes. In concert, the upstream toll-like receptor signaling and downstream NFκB signaling were inhibited at the multi-omics level. The inflammatory response was suppressed in the lungs, submandibular lymph nodes and mediastinal lymph nodes. We posited a causal chain of molecular mechanisms that indicated Y. pestis was probably able to impair host-mediated proteolysis activities and evade autophagosome capture by dysregulating both ubiquitin and microtubule networks in submandibular lymph nodes. Targeting these networks in a submandibular LN-specific and time-resolved fashion could be essential for development of the next generation therapeutics for pneumonic plague.


Assuntos
Pulmão/microbiologia , Linfonodos/microbiologia , Peste/genética , Primatas/genética , Primatas/microbiologia , Transcriptoma/genética , Yersinia pestis/fisiologia , Animais , Cercopithecus aethiops , Inflamação/genética , Inflamação/microbiologia , Masculino , Peste/microbiologia , Ubiquitina/genética
20.
J Biol Chem ; 294(15): 6113-6129, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30737286

RESUMO

Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases.


Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Substituição de Aminoácidos , Catálise , Domínio Catalítico , Humanos , Mutação de Sentido Incorreto , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
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