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1.
Ann Palliat Med ; 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33440964

RESUMO

BACKGROUND: High purity oxygen therapy has good clinical efficacy in the treatment of diabetic foot (DF), but its mechanism of promoting wound healing has been unclear. METHODS: Patients with DF were randomly divided into an experimental group and a control group. The experimental group was given local oxygen therapy (LOT) by a micro-oxygen therapy instrument, which administered uninterrupted >95% pure oxygen for 24 h at a flow rate of 3 mL/h. Six skin samples from the experimental group before and after treatment underwent RNA sequencing (RNA-seq), and the differentially expressed genes (DEGs) were screened. RESULTS: The clinical results showed that the mean wound healing time of the experimental group was 26 days (P<0.05); the healing area of the experimental group was 3.1-15.3 cm3 , with a mean of 8.8 cm3 , and that of the control group was 2.4-10.4 cm3 (P<0.05). LOT promoted the healing of DF wounds mainly through the tumor necrosis factor (TNF) signaling pathway and the apoptosis pathway. CONCLUSIONS: According to our results, LOT can promote DF healing mainly by inhibiting the local oxidative stress reaction of wound skin and by inhibiting the inflammatory and apoptotic pathways. The molecular markers and pathways screened warrant further study.

2.
mBio ; 11(5)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934080

RESUMO

Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSß), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

3.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
4.
Anal Chem ; 92(5): 3913-3922, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31992042

RESUMO

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.

5.
Adv Exp Med Biol ; 1206: 287-326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776992

RESUMO

Autophagy is an evolutionarily conserved lysosome-dependent intracellular degradation process that is essential for the maintenance of cellular homeostasis and adaptation to cellular stresses in eukaryotic cells. The most well-characterized type of autophagy, the macroautophagy, involves the progressive sequestration of cytoplasmic components into dedicated double-membraned vesicles called autophagosomes, which ultimately fuse with lysosomes to initiate the autophagic degradation of the sequestered cargo. In the past decade, our understanding of the molecular mechanism of macroautophagy has significantly evolved, with particular contributions from the biochemical and structural characterizations of autophagy-related proteins. In this chapter, we focus on some autophagy regulatory proteins involved in the macroautophagy pathway, summarize their currently known structures, and discuss their relevant molecular mechanisms from a perspective of structural biology.


Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Autofagossomos , Proteínas Relacionadas à Autofagia/química , Citosol , Lisossomos
6.
Nat Commun ; 10(1): 3459, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371777

RESUMO

Myosin VI plays crucial roles in diverse cellular processes. In autophagy, Myosin VI can facilitate the maturation of autophagosomes through interactions with Tom1 and the autophagy receptors, Optineurin, NDP52 and TAX1BP1. Here, we report the high-resolution crystal structure of the C-terminal cargo-binding domain (CBD) of Myosin VI in complex with Tom1, which elucidates the mechanistic basis underpinning the specific interaction between Myosin VI and Tom1, and uncovers that the C-terminal CBD of Myosin VI adopts a unique cargo recognition mode to interact with Tom1 for tethering. Furthermore, we show that Myosin VI can serve as a bridging adaptor to simultaneously interact with Tom1 and autophagy receptors through two distinct interfaces. In all, our findings provide mechanistic insights into the interactions of Myosin VI with Tom1 and relevant autophagy receptors, and are valuable for further understanding the functions of these proteins in autophagy and the cargo recognition modes of Myosin VI.


Assuntos
Citoesqueleto de Actina/metabolismo , Cadeias Pesadas de Miosina/química , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Autofagossomos/metabolismo , Autofagia/fisiologia , Proteínas de Ciclo Celular , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana Transportadoras , Modelos Moleculares , Proteínas de Neoplasias , Proteínas Nucleares , Ligação Proteica , Fator de Transcrição TFIIIA
8.
Proc Natl Acad Sci U S A ; 115(50): E11651-E11660, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30459273

RESUMO

NDP52 and TAX1BP1, two SKIP carboxyl homology (SKICH) domain-containing autophagy receptors, play crucial roles in selective autophagy. The autophagic functions of NDP52 and TAX1BP1 are regulated by TANK-binding kinase 1 (TBK1), which may associate with them through the adaptor NAP1. However, the molecular mechanism governing the interactions of NAP1 with NDP52 and TAX1BP1, as well as the effects induced by TBK1-mediated phosphorylation of NDP52 and TAX1BP1, remains elusive. Here, we report the atomic structures of the SKICH regions of NDP52 and TAX1BP1 in complex with NAP1, which not only uncover the mechanistic bases underpinning the specific interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1 but also reveal the binding mode of a SKICH domain. Moreover, we uncovered that the SKICH domains of NDP52 and TAX1BP1 share a general binding mode to interact with NAP1. Finally, we also evaluated the currently known TBK1-mediated phosphorylation sites in the SKICH domains of NDP52 and TAX1BP1 on the basis of their interactions with NAP1. In all, our findings provide mechanistic insights into the interactions of NAP1 with NDP52 and TAX1BP1, and are valuable for further understanding the functions of these proteins in selective autophagy.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Autofagia/fisiologia , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , tRNA Metiltransferases
9.
Sci Rep ; 8(1): 15238, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323290

RESUMO

Sixty norovirus outbreaks that occurred in Pudong District, Shanghai in 2017 and affected 959 people were summarised. Of the outbreaks, 29 (48.3%), 27 (45.0%), and 4 (6.7%) occurred in kindergartens, primary schools, and middle schools, respectively. Although the total number of outbreaks peaked in March (13/60, 21.7%), outbreaks in kindergartens and primary schools peaked in April (6/29, 20.7%) and March (8/27, 29.6%), respectively. Primary schools had the highest median number of cases per outbreak (19) and the highest proportion of cases (54.6%). The male-to-female case ratio differed among school classifications, with the highest male case ratio (69.2%) occurring in middle schools. Primary symptoms also differed across the school classifications. Molecular virology analysis showed that a single viral strain caused each outbreak at each school. In turn, 50.6, 28.8, and 20.6% of cases were infected by GII.4, GII.2, and GII.17, respectively. Vomiting was seen in 98.2, 97.3, and 88.6% of the subjects infected with noroviruses GII.17, GII.4, and GII.2, respectively, and nausea in 73.6, 43.9, and 39.0%. In conclusion, noroviruses mainly affect primary school and kindergarten students. GII.4, GII.2, and GII.17 are the main epidemic strains in the local area, and the primary symptoms differed by age and genotype.


Assuntos
Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite , Norovirus , Instituições Acadêmicas/estatística & dados numéricos , Adolescente , Fatores Etários , Criança , Pré-Escolar , China/epidemiologia , Surtos de Doenças , Feminino , Gastroenterite/diagnóstico , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Humanos , Masculino , Norovirus/genética , Norovirus/isolamento & purificação , Filogenia , RNA Viral/genética
10.
Cell ; 174(6): 1477-1491.e19, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30146158

RESUMO

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.


Assuntos
Apoptose , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Adulto , Idoso , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Axônios/metabolismo , Comportamento Animal , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Medula Espinal/metabolismo , Estaurosporina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
11.
Biochim Biophys Acta Mol Cell Res ; 1865(10): 1410-1422, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29981772

RESUMO

Linear ubiquitin chain is a latest discovered type of poly-ubiquitin chain that is broadly involved in innate immune and inflammatory pathways. Dysfunctions in its assembly, recognition or disassembly are intimately related with numerous immunodeficiency or autoimmune diseases. Our understanding of the molecular mechanism for linear ubiquitin chain formation, recognition and disassembly has being significantly evolved in recent years, with particular contribution from the biochemical and structural characterizations of related proteins. Here, we focus on the relevant proteins for the synthesis, recognition and digestion of linear ubiquitin chain, and review recent findings to summarize currently known molecular mechanism from a perspective of structural biology.

12.
J Mol Biol ; 430(18 Pt B): 3283-3296, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-29940186

RESUMO

TAX1BP1, a ubiquitin-binding adaptor, plays critical roles in the innate immunity and selective autophagy. During autophagy, TAX1BP1 may not only function as an autophagy receptor to recruit ubiquitylated substrates for autophagic degradation, but also serve as a Myosin VI cargo adaptor protein for mediating the maturation of autophagosome. However, the mechanistic basis underlying the specific interactions of TAX1BP1 with ubiquitin and Myosin VI remains elusive. Here, using biochemical, NMR and structural analyses, we elucidate the detailed binding mechanism and uncover the key determinants for the interaction between TAX1BP1 and ubiquitin. In addition, we reveal that both tandem zinc-fingers of TAX1BP1 and the conformational rigidity between them are required for the Myosin VI binding of TAX1BP1, and ubiquitin and Myosin VI are mutually exclusive in binding to TAX1BP1. Collectively, our findings provide mechanistic insights into the dual functions of TAX1BP1 in selective autophagy.


Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/química , Cadeias Pesadas de Miosina/química , Proteínas de Neoplasias/química , Ubiquitina/química , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Cadeias Pesadas de Miosina/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Ubiquitina/metabolismo
13.
Cell Chem Biol ; 25(6): 718-727.e3, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29657086

RESUMO

Here, we provide structural insights into PyrE3, a flavin-dependent [4 + 2] cyclase that catalyzes trans-decalin formation in the biosynthesis of pyrroindomycins. PyrE3 shares an architecture/domain organization head-to-tail similarity with the members of the family of para-hydroxybenzoate hydroxylase (pHBH)-fold monooxygenases, and possesses a flavin adenine dinucleotide (FAD)-binding domain, a middle domain, and a C-terminal thioredoxin-like domain. The FAD-binding domain forms a central hub of the protein structure, and binds with FAD in a "closed" conformation of pHBH-fold family monooxygenases known for their highly dynamic catalytic processes. FAD plays an essential structural role in PyrE3, where it is amenable to redox change; however, redox change has little effect on [4 + 2] cyclization activity. PyrE3 appears to selectively accommodate a tetramate-containing, linear polyene intermediate in a highly positively charged pocket, which is located at the interface between the FAD-binding domain and the middle domain, and can accelerate trans-decalin formation likely through an endo-selective [4 + 2] transition state.


Assuntos
Dinitrocresóis/metabolismo , Macrolídeos/metabolismo , Oxigenases de Função Mista/metabolismo , Naftalenos/metabolismo , Biocatálise , Cristalografia por Raios X , Dinitrocresóis/química , Macrolídeos/química , Oxigenases de Função Mista/química , Modelos Moleculares , Estrutura Molecular , Naftalenos/química
14.
Front Mol Neurosci ; 11: 64, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29541021

RESUMO

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

15.
Gut Pathog ; 10: 7, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29483945

RESUMO

Background: Norovirus (NoV), a member of the Caliciviridae, is now recognized as the leading cause of acute gastroenteritis (AGE) worldwide. Globally, the GII.4 Sydney_2012 variant has predominated in NoV-related AGE since 2012, although the novel variant GII.17 has also been reported as responsible for gastroenteritis outbreaks in East Asia since 2014. This study aimed to disclose the recent genotype patterns of NoV genogroup II (GII) presenting in AGE patients in Pudong New Area of Shanghai through a laboratory-based syndromic surveillance system. The study further aimed to delineate the predominant strains circulating in the population. Methods: Pudong New Area is located in eastern Shanghai and covers 20.89% of the Shanghai population. The laboratory-based syndromic surveillance system is composed of 12 sentinel hospitals among the 68 general hospitals in this area. AGE patients who sought medical care were sampled following an AGE surveillance protocol. Stool samples were collected from participating patients, and a standardized questionnaire was given to each patient by trained nurses to gain information on the disease profiles and demographics of the patients. Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to screen the GI nd GII NoV and RT-PCR was used to amplify NoV GII partial capsid protein open reading frame 2 (ORF2). NoV Genotyping Tool (version 1.0, RIVM, MA Bilthoven, Netherlands) was used for genotyping, and a phylogenetic analysis was conducted by MEGA 7.0. Results: During 2014-2016, among the 2069 virus-infected AGE cases, 65.88% were caused by NoV. NoV-AGE occurred most frequently in the periods from October to March. The patients with more severe diarrheal symptoms and vomiting were more likely to be infected by NoV. The main genotypes were GII.17 (44.69%) and GII.4 (39.26%), which dominated the NoV-AGE epidemics jointly or in turn, whereas a slight increase in GII.2 was observed beginning in May 2016. The GII.17 strains tended to cluster more with the Hu/JP/2014/GII.P17_GII.17/Kawasaki323 variants, representing novel prevalent strains. Among the GII.4 strains, the GII.4 Sydney_2012 variant was still the predominant strain. Conclusions: NoV GII has become the main cause of virus-infected AGE in Pudong New Area, Shanghai. The predominant genotypes of NoV GII were GII.17 and GII.4. Comprehensive laboratory-based surveillance is important for clinical diagnosis and treatment. Identification of emerging new genotypes is also crucial for the prevention and control of NoV-infected AGE.

16.
Autophagy ; 14(1): 66-79, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29394115

RESUMO

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.


Assuntos
Autofagia , Doenças Neurodegenerativas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Ubiquitina/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ciclo Celular , Glaucoma/genética , Glaucoma/metabolismo , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteínas de Membrana Transportadoras , Mutação , Doenças Neurodegenerativas/genética , Fosforilação , Agregados Proteicos , Ligação Proteica , Fator de Transcrição TFIIIA/genética
17.
Cell Rep ; 21(1): 27-36, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978479

RESUMO

The linear ubiquitin chain assembly complex (LUBAC) is the sole identified E3 ligase complex that catalyzes the formation of linear ubiquitin chain, and it is composed of HOIP, HOIL-1L, and SHARPIN. The E3 activity of HOIP can be effectively activated by HOIL-1L or SHARPIN, deficiency of which leads to severe immune system disorders. However, the underlying mechanism governing the HOIP-SHARPIN interaction and the SHARPIN-mediated activation of HOIP remains elusive. Here, we biochemically and structurally demonstrate that the UBL domain of SHARPIN specifically binds to the UBA domain of HOIP and thereby associates with and activates HOIP. We further uncover that SHARPIN and HOIL-1L can separately or synergistically bind to distinct sites of HOIP UBA with induced allosteric effects and thereby facilitate the E2 loading of HOIP for its activation. Thus, our findings provide mechanistic insights into the assembly and activation of LUBAC.


Assuntos
Fatores de Transcrição/química , Ubiquitina-Proteína Ligases/química , Ubiquitinas/química , Substituição de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo
18.
Genes Dev ; 31(11): 1162-1176, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701375

RESUMO

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.


Assuntos
Proteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/genética , Animais , Apoptose/genética , Células Cultivadas , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases/genética , Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/genética
19.
Genes Dev ; 31(10): 1024-1035, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28619731

RESUMO

Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of ß-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of ß-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/ß-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/ß-catenin pathway.


Assuntos
Estabilidade Enzimática/genética , Tanquirases/metabolismo , Ubiquitina Tiolesterase/metabolismo , Via de Sinalização Wnt/fisiologia , Repetição de Anquirina , Proteína Axina/metabolismo , Linhagem Celular , Cristalografia por Raios X , Células HCT116 , Células HEK293 , Humanos , Mutação , Ligação Proteica , Tanquirases/química , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genética , Via de Sinalização Wnt/genética
20.
J Infect Public Health ; 10(6): 725-729, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28196637

RESUMO

More than 30 residents and nursing assistants in a geriatric nursing hospital developed acute gastroenteritis from December 7th to December 18th, 2014 in Shanghai, China. An immediate epidemiological investigation was conducted to identify the etiological agent of the outbreak, mode of transmission and the risk factors. Cases were investigated according to an epidemiological questionnaire. Samples from cases, highly transmissible environmental surfaces and drinking water were collected for pathogens detection. A retrospective cohort study was conducted to explore the transmission mode. A total of 34 cases were affected in this acute gastroenteritis outbreak, including 23 residents, 9 nursing assistants and 2 doctors. 13 out of 30 samples were positive for GII.17 norovirus, no other pathogen was detected. Nursing assistants who developed gastroenteritis symptoms had a higher attack rate in residents they cared than those who did not develop any gastroenteritis symptoms (p<0.001). The acute gastroenteritis outbreak was caused by GII.17 norovirus. Person-to-person close contact and contaminated environmental surfaces were the probable transmission route. Nursing assistants were considered to play an important role in the secondary spread of norovirus. The poor medical skill and personal hygiene habits of nursing assistants in China should be paid attention and improved urgently which is critically important to prevent hospital infections.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Gastroenterite/epidemiologia , Genótipo , Norovirus/classificação , Enfermeiras e Enfermeiros , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , China , Infecção Hospitalar/transmissão , Infecção Hospitalar/virologia , Transmissão de Doença Infecciosa , Microbiologia Ambiental , Fezes/microbiologia , Feminino , Gastroenterite/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Norovirus/genética , Norovirus/isolamento & purificação , Casas de Saúde , Estudos Retrospectivos
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