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1.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638625

RESUMO

Glycosyltransferase OGT catalyzes the conjugation of O-linked ß-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin-proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.


Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Papillomaviridae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação/fisiologia
2.
Nat Commun ; 12(1): 5772, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599178

RESUMO

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.


Assuntos
Adenosina Trifosfatases/metabolismo , Anti-Infecciosos/metabolismo , Citocinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Células A549 , Animais , Enterovirus/fisiologia , Células HEK293 , Células HeLa , Herpesvirus Humano 1/fisiologia , Humanos , Interferon Tipo I/metabolismo , Gotículas Lipídicas/metabolismo , Listeria monocytogenes/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Ligação Proteica , Multimerização Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Células THP-1 , Ubiquitina/metabolismo
3.
Nat Commun ; 12(1): 5939, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642328

RESUMO

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/classificação , Proteínas de Choque Térmico HSP70/genética , Humanos , Cinética , Proteína NEDD8/química , Proteína NEDD8/genética , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/genética , Ubiquitinação
4.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638579

RESUMO

Parkinson's disease (PD) is a degenerative disease that can cause motor, cognitive, and behavioral disorders. The treatment strategies being developed are based on the typical pathologic features of PD, including the death of dopaminergic (DA) neurons in the substantia nigra of the midbrain and the accumulation of α-synuclein in neurons. Peiminine (PMN) is an extract of Fritillaria thunbergii Miq that has antioxidant and anti-neuroinflammatory effects. We used Caenorhabditis elegans and SH-SY5Y cell models of PD to evaluate the neuroprotective potential of PMN and address its corresponding mechanism of action. We found that pretreatment with PMN reduced reactive oxygen species production and DA neuron degeneration caused by exposure to 6-hydroxydopamine (6-OHDA), and therefore significantly improved the DA-mediated food-sensing behavior of 6-OHDA-exposed worms and prolonged their lifespan. PMN also diminished the accumulation of α-synuclein in transgenic worms and transfected cells. In our study of the mechanism of action, we found that PMN lessened ARTS-mediated degradation of X-linked inhibitor of apoptosis (XIAP) by enhancing the expression of PINK1/parkin. This led to reduced 6-OHDA-induced apoptosis, enhanced activity of the ubiquitin-proteasome system, and increased autophagy, which diminished the accumulation of α-synuclein. The use of small interfering RNA to down-regulate parkin reversed the benefits of PMN in the PD models. Our findings suggest PMN as a candidate compound worthy of further evaluation for the treatment of PD.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cevanas/farmacologia , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Degeneração Neural/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Substância Negra/metabolismo , Ubiquitina/metabolismo
5.
Phytomedicine ; 92: 153763, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34601222

RESUMO

BACKGROUND: Misfolded proteins are formed in the endoplasmic reticulum (ER) due to diverse stimuli including oxidant production, calcium disturbance, and inflammatory factors. Accumulation of these non-native proteins in the ER evokes cellular stress involving the activation of unfolded protein response (UPR) and the execution of ER-associated degradation (ERAD). Naturally-occurring plant compounds are known to interfere with UPR due to their antioxidant and anti-inflammatory activities, leading to inhibition of ER stress. However, there are few studies dealing with the protective effects of natural compounds on the functionality of ERAD. PURPOSE: The current study examined whether asaronic acid enhanced ubiquitin-proteasomal degradation in J774A.1 murine macrophages exposed to 7ß-hydroxycholesterol, a risk factor for atherosclerosis. Asaronic acid (2,4,5-trimethoxybenzoic acid), identified as one of purple perilla constituents, has anti-diabetic and anti-inflammatory effects. Little is known regarding the effects of asaronic acid on the ERAD process and the ubiquitin-proteasomal degradation. METHODS AND RESULTS: Murine macrophages were incubated with 28 µM 7ß-hydroxycholesterol in absence and presence of 1-20 µΜ asaronic acid for up to 24 h. Nontoxic asaronic acid in macrophage diminished the activation of the ER stress sensors of ATF6, IRE1 and PERK stimulated by 7ß-hydroxycholesterol. This methoxybenzoic acid down-regulated the oxysterol-induced expression of EDEM1, OS9, Sel1L-Hrd1 and p97/VCP1, all required for the recognition, recruitment and dislocation of misfolded proteins. On the other hand, asaronic acid enhanced the ubiquitin-proteasomal degradation of non-native proteins dislocated to the cytosol by 7ß-hydroxycholesterol, which entailed the induction of the chaperones of Hsp70 and CHIP and the increased colocalization of ubiquitin and proteasomes. Taken together, asaronic acid attenuated the induction of the UPR-associated sensors and the dislocation-linked transmembrane components in the ER. Conversely, this compound enhanced the proteasomal degradation of dislocated non-native proteins in concert with the chaperones of Hsp70 and CHIP through ubiquitination. CONCLUSION: These observations demonstrate that asaronic acid may be a potent atheroprotective agent as a natural chaperone targeting ER stress-associated macrophage injury.


Assuntos
Hidroxicolesteróis , Ubiquitina , Animais , Estresse do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Macrófagos , Camundongos
6.
Phytomedicine ; 93: 153799, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34715511

RESUMO

BACKGROUND: Natural medicines have a long history in the prevention and treatment of various diseases in East Asian region, especially in China. Modern research has proved that the pharmacological effects of numerous natural medicines involve the participation of ubiquitin proteasome system (UPS). UPS can degrade the unwanted and damaged proteins widely distributed in the nucleus and cytoplasm of various eukaryotes. PURPOSE: The objective of the present study was to review and discuss the regulatory effects of natural products and extracts on proteasome components, which may help to find new proteasome regulators for drug development and clinical applications. METHODS: The related information was compiled using the major scientific databases, such as CNKI, Elsevier, ScienceDirect, PubMed, SpringerLink, Wiley Online, and GeenMedical. The keywords "natural product" and "proteasome" were applied to extract the literature. Nature derived extracts, compounds and their derivatives involved in proteasome regulation were included, and the publications related to synthetic proteasome agents were excluded. RESULTS: The pharmacological effects of more than 80 natural products and extracts derived from phytomedicines related to the proteasome regulation were reviewed. These natural products were classified according to their chemical properties. We also summarized some laws of action of natural products as proteasome regulators in the treatment of diseases, and listed the action characteristics of the typical natural products. CONCLUSION: Natural products derived from nature can induce the degradation of damaged proteins through UPS or act as regulators to directly regulate the activity of proteasome. But few proteasome modulators are applied clinically. Summary of known rules for proteasome modulators will contribute to discover, modify and synthesize more proteasome modulators for clinical applications.


Assuntos
Produtos Biológicos , Complexo de Endopeptidases do Proteassoma , Produtos Biológicos/farmacologia , China , Citoplasma , Ubiquitina
7.
Nat Commun ; 12(1): 5913, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625556

RESUMO

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.


Assuntos
Endopeptidases/metabolismo , Queratinócitos/metabolismo , Pele/metabolismo , Animais , Morte Celular/genética , Citocinas/metabolismo , Endopeptidases/genética , Proteína de Domínio de Morte Associada a Fas , Técnicas de Introdução de Genes , Homeostase , Inflamação/patologia , Interferon Tipo I , Interleucina-1beta , Camundongos , Necroptose , Fragmentos de Peptídeos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Pele/patologia , Células-Tronco/metabolismo , Análise de Sistemas , Ubiquitina/metabolismo
8.
Cell Physiol Biochem ; 55(S4): 68-95, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34523304

RESUMO

Protein homeostasis strongly depends on the targeted and selective removal of unneeded or flawed proteins, of protein aggregates, and of damaged or excess organelles by the two main intracellular degradative systems, namely the ubiquitin proteasomal system (UPS) and the autophagosomal lysosomal system. Despite representing completely distinct mechanisms of degradation, which underlie differing regulatory mechanisms, growing evidence suggests that the UPS and autophagy strongly interact especially in situations of overwhelming and impairment, and that both are involved in podocyte proteostasis and in the pathogenesis of podocyte injury. The differential impact of autophagy and the UPS on podocyte biology and on podocyte disease development and progression is not understood. Recent advances in understanding the role of the UPS and autophagy in podocyte biology are reviewed here.


Assuntos
Autofagia , Nefropatias , Podócitos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/patologia , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/fisiopatologia , Lisossomos/metabolismo , Lisossomos/patologia , Podócitos/metabolismo , Podócitos/patologia
9.
Nat Cell Biol ; 23(9): 978-991, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497368

RESUMO

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Poliubiquitina/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Genes Supressores de Tumor/fisiologia , Humanos , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo
10.
Chem Commun (Camb) ; 57(74): 9438-9441, 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528945

RESUMO

Protein post-translational modifications are involved in essentially all aspects of cellular signaling. Their dynamic nature and the difficulties in installing them using enzymatic approaches limits their direct study in human cells. Reported herein is the first synthesis, delivery and cellular study of a stable phosphoubiquitin probe. Our results compare Parkin's substrate preference during mitophagy via direct visualization of a phosphorylated ubiquitin probe in the cellular environment.


Assuntos
Sondas Moleculares/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Humanos , Sondas Moleculares/química , Estrutura Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Ubiquitina/química
11.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502538

RESUMO

The ubiquitin system, present in all eukaryotes, contributes to regulating multiple types of cellular protein processes such as cell signaling, cell cycle, and receptor trafficking, and it affects the immune response. In most types of cancer, unusual events in ubiquitin-mediated signaling pathway modulation can lead to a variety of clinical outcomes, including tumor formation and metastasis. Similarly, ubiquitination acts as a core component, which contributes to the alteration of cell signaling activity, dictating biosignal turnover and protein fates. As lung cancer acquires the most commonly mutated proteins, changes in the ubiquitination of the proteins contribute to the development of lung cancer. Various inhibitors targeting the ubiquitin system have been developed for clinical applications in lung cancer treatment. In this review, we summarize the current research advances in therapeutics for lung cancer by targeting the ubiquitin system.


Assuntos
Bortezomib/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina/metabolismo , Ubiquitinação , Animais , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Inibidores de Proteassoma/uso terapêutico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo
12.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502365

RESUMO

Plant U-box armadillo repeat (PUB-ARM) ubiquitin (Ub) ligases have important functions in plant defense through the ubiquitination of target proteins. Defense against pathogens involves vesicle trafficking and the formation of extracellular vesicles. The PUB-ARM protein SENESCENCE ASSOCIATED UBIQUITIN E3 LIGASE1 (SAUL1) can form patches at the plasma membrane related to tethering multi-vesicular bodies (MVBs) to the plasma membrane. We uncovered the structure of a full-length plant ubiquitin ligase and the structural requirements of SAUL1, which are crucial for its function in patch formation. We resolved the structure of SAUL1 monomers by small-angle X-ray scattering (SAXS). The SAUL1 model showed that SAUL1 consists of two domains: a domain containing the N-terminal U-box and armadillo (ARM) repeats and the C-terminal ARM repeat domain, which includes a positively charged groove. We showed that all C-terminal ARM repeats are essential for patch formation and that this function requires arginine residue at position 736. By applying SAXS to polydisperse SAUL1 systems, the oligomerization of SAUL1 is detectable, with SAUL1 tetramers being the most prominent oligomers at higher concentrations. The oligomerization domain consists of the N-terminal U-box and some N-terminal ARM repeats. Deleting the U-box resulted in the promotion of the SAUL1 tethering function. Our findings indicate that structural changes in SAUL1 may be fundamental to its function in forming patches at the plasma membrane.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestrutura , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Domínios Proteicos/genética , Transporte Proteico , Espalhamento a Baixo Ângulo , Ubiquitina/metabolismo , Ubiquitinação , Difração de Raios X/métodos
13.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576118

RESUMO

Rett Syndrome (RTT) is an X linked neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, resulting in severe cognitive and physical disabilities. Despite an apparent normal prenatal and postnatal development period, symptoms usually present around 6 to 18 months of age. Little is known about the consequences of MeCP2 deficiency at a molecular and cellular level before the onset of symptoms in neural cells, and subtle changes at this highly sensitive developmental stage may begin earlier than symptomatic manifestation. Recent transcriptomic studies of patient induced pluripotent stem cells (iPSC)-differentiated neurons and brain organoids harbouring pathogenic mutations in MECP2, have unravelled new insights into the cellular and molecular changes caused by these mutations. Here we interrogated transcriptomic modifications in RTT patients using publicly available RNA-sequencing datasets of patient iPSCs harbouring pathogenic mutations and healthy control iPSCs by Weighted Gene Correlation Network Analysis (WGCNA). Preservation analysis identified core gene pathways involved in translation, ribosomal function, and ubiquitination perturbed in some MECP2 mutant iPSC lines. Furthermore, differential gene expression of the parental fibroblasts and iPSC-derived neurons revealed alterations in genes in the ubiquitination pathway and neurotransmission in fibroblasts and differentiated neurons respectively. These findings might suggest that global translational dysregulation and proteasome ubiquitin function in Rett syndrome begins in progenitor cells prior to lineage commitment and differentiation into neural cells.


Assuntos
Redes Reguladoras de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/genética , Síndrome de Rett/genética , Ubiquitina/metabolismo , Análise por Conglomerados , Bases de Dados Genéticas , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neurônios/metabolismo , Análise de Componente Principal , Domínios Proteicos , Ubiquitina/genética
14.
J Vis Exp ; (174)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34515691

RESUMO

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Bacteriófago M13 , Técnicas de Visualização da Superfície Celular , Humanos , Biblioteca de Peptídeos , Conformação Proteica , Ubiquitina/genética
15.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502293

RESUMO

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.


Assuntos
Proteína Semelhante a ELAV 2/metabolismo , Regulação da Expressão Gênica , Estresse Fisiológico , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteína Semelhante a ELAV 2/genética , Humanos , Ubiquitinas/genética
16.
Nat Commun ; 12(1): 5399, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518535

RESUMO

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.


Assuntos
Redes Neurais de Computação , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação , Linhagem Celular Tumoral , Células HCT116 , Humanos , Células Jurkat , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo , Ubiquitina/metabolismo
17.
Nat Commun ; 12(1): 5337, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504101

RESUMO

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.


Assuntos
Proteínas 14-3-3/genética , Proteínas Fetais/genética , Linfócitos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/genética , Ubiquitina/genética , Proteínas 14-3-3/metabolismo , Células A549 , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas Fetais/antagonistas & inibidores , Proteínas Fetais/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Anal Chem ; 93(37): 12748-12757, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34494821

RESUMO

Native electrospray ionization (ESI)-mass spectrometry (MS) is widely used for the detection and characterization of multi-protein complexes. A well-known problem with this approach is the possible occurrence of nonspecific protein clustering in the ESI plume. This effect can distort the results of binding affinity measurements, and it can even generate gas-phase complexes from proteins that are strictly monomeric in bulk solution. By combining experiments and molecular dynamics (MD) simulations, the current work for the first time provides detailed insights into the ESI clustering of proteins. Using ubiquitin as a model system, we demonstrate how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific clusters (e.g., dimers or trimers) via solvent evaporation to dryness. These events are in line with earlier proposals, according to which protein clustering is associated with the charged residue model (CRM). MD simulations on cytochrome c (which carries a large intrinsic positive charge) confirmed the viability of this CRM avenue. In addition, the cytochrome c data uncovered an alternative mechanism where protein-protein contacts were formed early within ESI droplets, followed by cluster ejection from the droplet surface. This second pathway is consistent with the ion evaporation model (IEM). The observation of these IEM events for large protein clusters is unexpected because the IEM has been thought to be associated primarily with low-molecular-weight analytes. In all cases, our MD simulations produced protein clusters that were stabilized by intermolecular salt bridges. The MD-generated charge states agreed with experiments. Overall, this work reveals that ESI-induced protein clustering does not follow a tightly orchestrated pathway but can proceed along different avenues.


Assuntos
Simulação de Dinâmica Molecular , Espectrometria de Massas por Ionização por Electrospray , Proteínas , Solventes , Ubiquitina
19.
Nat Commun ; 12(1): 5212, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471133

RESUMO

The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62-ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.


Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo
20.
Cell Rep ; 36(13): 109754, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34547223

RESUMO

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/ultraestrutura , SARS-CoV-2/genética , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , COVID-19/genética , COVID-19/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/genética , Células HEK293 , Humanos , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Ligação Proteica/genética , SARS-CoV-2/metabolismo , Especificidade por Substrato/genética , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
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