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1.
Nat Commun ; 12(1): 302, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436591

RESUMO

Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.


Assuntos
Doenças Autoimunes/enzimologia , Doenças Autoimunes/patologia , Granzimas/antagonistas & inibidores , Granzimas/metabolismo , Animais , Autoantígenos/metabolismo , Vesícula , Quimiocina CXCL2/metabolismo , Fatores Quimiotáticos/farmacologia , Modelos Animais de Doenças , Epidermólise Bolhosa/enzimologia , Epidermólise Bolhosa/patologia , Humanos , Inflamação/patologia , Integrina alfa6/metabolismo , Interleucina-8/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Colágenos não Fibrilares/metabolismo , Penfigoide Bolhoso/enzimologia , Penfigoide Bolhoso/patologia , Índice de Gravidade de Doença
2.
Anticancer Res ; 41(2): 1089-1099, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517320

RESUMO

BACKGROUND/AIM: Prognosis plays a vital role in head and neck squamous cell carcinoma (HNSCC) patient management and decision-making. This study aimed to identify the role of BP180 as a prognostic factor in HNSCC. PATIENTS AND METHODS: Protein expression of bullous pemphigoid antigen II (BP180) was verified by immunohistochemistry (IHC) in a tissue microarray study of 202 cases. RESULTS: IHC analysis revealed that protein expression of BP180 among HNSCC patients differed significantly in the presence and absence of neural invasion, and according to T status in laryngeal and pharyngeal cancer subgroups. Overall survival and multivariate analysis showed that positive BP180-IHC and advanced clinical stage were significant independent positive predictors of mortality in HNSCC patients. In addition, in the oral cancer subgroup, independent positive predictors were positive BP180-IHC, advanced N status and neural invasion. In laryngeal and pharyngeal cancer subgroups, predictors were positive BP180-IHC and advanced clinical stage. CONCLUSION: BP180 is a prognostic factor in head and neck squamous cell carcinoma.


Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Colágenos não Fibrilares/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Tomada de Decisão Clínica , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Análise de Sobrevida , Análise Serial de Tecidos
3.
Proc Natl Acad Sci U S A ; 117(36): 22341-22350, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32855302

RESUMO

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.


Assuntos
Anticorpos , Diversidade de Anticorpos/genética , Autoantígenos , Rearranjo Gênico do Linfócito B/genética , Mutação/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Autoanticorpos/química , Autoanticorpos/genética , Autoanticorpos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Regiões Determinantes de Complementaridade/genética , Imunidade Humoral/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Hipermutação Somática de Imunoglobulina/genética
4.
PLoS One ; 15(8): e0237907, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822386

RESUMO

Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cut-off of 2-fold. These included pathways involving transcription factors associated with the regulation of pro-inflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up- or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noise-induced hearing loss under conditions that do not cause permanent hearing loss in age/strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.


Assuntos
Regulação da Expressão Gênica/genética , Perda Auditiva Provocada por Ruído/metabolismo , Nefrite Hereditária/metabolismo , Estria Vascular/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Matriz Extracelular/metabolismo , Feminino , Glucose/genética , Glucose/metabolismo , Perda Auditiva Provocada por Ruído/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , RNA-Seq , Transdução de Sinais/genética , Estria Vascular/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Regulação para Cima
5.
Nat Commun ; 11(1): 3904, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764536

RESUMO

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.


Assuntos
Autoantígenos/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Autoantígenos/genética , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Proteína Supressora de Tumor p53/genética , Fosfatases cdc25/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Antivirais/farmacologia , Genoma Bacteriano , Macrolídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
7.
Curr Opin Endocrinol Diabetes Obes ; 27(4): 240-247, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32618636

RESUMO

PURPOSE OF REVIEW: The role of T cells specific for islet autoantigens is proven in pathogenesis of type 1 diabetes. Recently, there has been rapid expansion in the number of T-cell subsets identified, this has coincided with an increase in the repertoire of reported islet antigens mainly through the discovery of novel epitopes. A discussion of how these marry together is now warranted and timely. RECENT FINDINGS: In this review, we will discuss the autoreactivity against neo-epitopes. We then explore the growing array of T-cell subsets for both CD4 T cells, including follicular and peripheral T helper cells, and CD8 T cells, discussing evolution from naïve to exhausted phenotypes. Finally, we detail how subsets correlate with disease stage and loss of ß-cell function and are impacted by immunotherapy. SUMMARY: The expanding list of T-cell subsets may be potentially encouraging in terms of elucidating disease mechanisms and have a role as biomarkers for disease progression. Furthermore, T-cell subsets can be used in stratifying patients for clinical trials and for monitoring immunotherapy outcomes. However, the definition of subsets needs to be refined in order to ensure that there is a uniform approach in designating T-cell subset attributes that is globally applied.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Subpopulações de Linfócitos T/fisiologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Ilhotas Pancreáticas/imunologia , Subpopulações de Linfócitos T/imunologia
8.
Biochem Biophys Res Commun ; 529(2): 251-256, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32703419

RESUMO

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.


Assuntos
Autoantígenos/metabolismo , Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Técnicas In Vitro , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Ligação Proteica , Estabilidade Proteica , Transporte Proteico
9.
Nat Commun ; 11(1): 2818, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499524

RESUMO

In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. Here, we show that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.


Assuntos
Adenosina Trifosfatases/metabolismo , Autoantígenos/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/química , Histona Desacetilase 1/metabolismo , Nucleossomos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Drosophila melanogaster , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Histonas/química , Ovário/metabolismo , Proteínas Inibidoras de STAT Ativados
10.
PLoS Biol ; 18(6): e3000679, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555591

RESUMO

Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts.


Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Grânulos Citoplasmáticos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose , Fenótipo , Domínios Proteicos , Multimerização Proteica , Reprodutibilidade dos Testes
11.
Nat Commun ; 11(1): 2859, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503973

RESUMO

Mature double negative (DN) T cells are a population of αß T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.


Assuntos
Autoantígenos/imunologia , Interleucina-17/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoantígenos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Tolerância Imunológica , Antígeno Ki-67/imunologia , Antígeno Ki-67/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo
12.
Prostate ; 80(9): 715-726, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32364250

RESUMO

BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Catepsina L/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Masculino , Espectrometria de Massas , Invasividade Neoplásica , Colágenos não Fibrilares/metabolismo , Células PC-3 , Proteômica/métodos , Esferoides Celulares
13.
PLoS Biol ; 18(5): e3000746, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453802

RESUMO

Members of the Tre2-Bub2-Cdc16 (TBC) family often function to regulate membrane trafficking and to control signaling transductions pathways. As a member of the TBC family, TBC1D23 is critical for endosome-to-Golgi cargo trafficking by serving as a bridge between Golgi-bound golgin-97/245 and the WASH/FAM21 complex on endosomal vesicles. However, the exact mechanisms by which TBC1D23 regulates cargo transport are poorly understood. Here, we present the crystal structure of the N-terminus of TBC1D23 (D23N), which consists of both the TBC and rhodanese domains. We show that the rhodanese domain is unlikely to be an active sulfurtransferase or phosphatase, despite containing a putative catalytic site. Instead, it packs against the TBC domain and forms part of the platform to interact with golgin-97/245. Using the zebrafish model, we show that impacting golgin-97/245-binding, but not the putative catalytic site, impairs neuronal growth and brain development. Altogether, our studies provide structural and functional insights into an essential protein that is required for organelle-specific trafficking and brain development.


Assuntos
Autoantígenos/metabolismo , Encéfalo/embriologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Tiossulfato Sulfurtransferase/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Animais , Escherichia coli , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/isolamento & purificação , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Conformação Proteica , Domínios Proteicos , Peixe-Zebra
14.
Nat Rev Rheumatol ; 16(6): 301-315, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341463

RESUMO

Peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) owing to their ability to generate citrullinated proteins - the hallmark autoantigens of RA. Of the five PAD enzyme isoforms, PAD2 and PAD4 are the most strongly implicated in RA at both genetic and cellular levels, and PAD inhibitors have shown therapeutic efficacy in mouse models of inflammatory arthritis. PAD2 and PAD4 are additionally targeted by autoantibodies in distinct clinical subsets of patients with RA, suggesting anti-PAD antibodies as possible biomarkers for RA diagnosis and prognosis. This Review weighs the evidence that supports a pathogenic role for PAD enzymes in RA as both promoters and targets of the autoimmune response, as well as discussing the mechanistic and therapeutic implications of these findings in the wider context of RA pathogenesis. Understanding the origin and consequences of dysregulated PAD enzyme activity and immune responses against PAD enzymes will be important to fully comprehend the pathogenic mechanisms involved in this disease and for the development of novel strategies to treat and prevent RA.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Citrulinação , Reações Cruzadas , Predisposição Genética para Doença , Humanos , Doenças Pulmonares Intersticiais/imunologia , Proteína-Arginina Desiminase do Tipo 2/genética , Proteína-Arginina Desiminase do Tipo 2/imunologia , Proteína-Arginina Desiminase do Tipo 3/imunologia , Proteína-Arginina Desiminase do Tipo 4/genética , Proteína-Arginina Desiminase do Tipo 4/imunologia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Índice de Gravidade de Doença
15.
Scand J Immunol ; 91(6): e12888, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32281665

RESUMO

We propose a framework to explain how T cells achieve specificity and sensitivity, how the affinity of the TcR peptide/MHC interaction controls positive and negative thymic selection and mature T cell survival, and whether antigen-dependent activation and inactivation takes place. Two distinct types of signalling can lead to mature T cell multiplication. One requires the TcR to recognize with a certain affinity an antigen-derived peptide, an agonist peptide, bound to an MHC molecule. The other, the tonic signal, leads to naïve T cell survival and modest proliferation if the T cell successfully competes for endogenous, self-peptide/MHC ligands, involving lower affinity TCR/ligand interactions. Many suggest lymphopenia contributes to autoimmunity by increasing the strength of TcR-tonic signalling, and so activation of anti-self T cells. We suggest T cell activation requires antigen-mediated cooperation between T cells. Increased tonic signalling under lymphopenic conditions facilitates T cell proliferation and so antigen-dependent cooperation and activation of anti-self T cells.


Assuntos
Linfopenia/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologia , Animais , Apresentação do Antígeno , Autoantígenos/imunologia , Autoantígenos/metabolismo , Autoimunidade , Comunicação Celular , Diferenciação Celular , Sobrevivência Celular , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ativação Linfocitária , Modelos Imunológicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
16.
Oncogene ; 39(17): 3522-3540, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32111984

RESUMO

Metastasis is a major cause of high recurrence and poor survival of patients with colorectal cancer (CRC), although the mechanisms associated with this process remain poorly understood. In this study, we report a novel mechanism by which SOX13 promotes CRC metastasis by transactivating SNAI2 and c-MET. SOX13 overexpression was significantly correlated with more aggressive clinicopathological features of CRC and indicated poor prognosis in two independent cohorts of CRC patients (cohort I, n = 363; cohort II, n = 390). Overexpression of SOX13-promoted CRC migration, invasion, and metastasis, whereas SOX13 downregulation caused the opposite effects. Further mechanistic investigation identified SNAI2 and MET as important target genes of SOX13 using serial deletion and site-directed mutagenesis luciferase reporter and chromatin immunoprecipitation (ChIP) assays, as well as functional complementation analyses. In addition, SOX13 was shown to be a direct target of HGF/STAT3 signaling, and the c-MET inhibitor crizotinib blocked the HGF/STAT3/SOX13/c-MET axis, significantly inhibiting SOX13-mediated CRC migration, invasion and metastasis. Moreover, in clinical CRC tissues, SOX13 expression was positively correlated with the expression of SNAI2, c-MET, and HGF. CRC patients with positive coexpression of SOX13/SNAI2, SOX13/c-MET, or HGF/SOX13 exhibited a worse prognosis. In summary, SOX13 is a promising prognostic biomarker in patients with CRC, and blocking the HGF/STAT3/SOX13/c-MET axis with crizotinib could be a new therapeutic strategy to prevent SOX13-mediated CRC metastasis.


Assuntos
Autoantígenos/metabolismo , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-met/biossíntese , Fatores de Transcrição SOXD/metabolismo , Fatores de Transcrição da Família Snail/biossíntese , Ativação Transcricional , Animais , Autoantígenos/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-met/genética , Fatores de Transcrição SOXD/genética , Fatores de Transcrição da Família Snail/genética
17.
BMC Bioinformatics ; 21(Suppl 2): 78, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32164523

RESUMO

BACKGROUND: Finding the tumor location in the prostate is an essential pathological step for prostate cancer diagnosis and treatment. The location of the tumor - the laterality - can be unilateral (the tumor is affecting one side of the prostate), or bilateral on both sides. Nevertheless, the tumor can be overestimated or underestimated by standard screening methods. In this work, a combination of efficient machine learning methods for feature selection and classification are proposed to analyze gene activity and select them as relevant biomarkers for different laterality samples. RESULTS: A data set that consists of 450 samples was used in this study. The samples were divided into three laterality classes (left, right, bilateral). The aim of this work is to understand the genomic activity in each class and find relevant genes as indicators for each class with nearly 99% accuracy. The system identified groups of differentially expressed genes (RTN1, HLA-DMB, MRI1) that are able to differentiate samples among the three classes. CONCLUSION: The proposed method was able to detect sets of genes that can identify different laterality classes. The resulting genes are found to be strongly correlated with disease progression. HLA-DMB and EIF4G2, which are detected in the set of genes can detect the left laterality, were reported earlier to be in the same pathway called Allograft rejection SuperPath.


Assuntos
Regulação Neoplásica da Expressão Gênica , Aprendizado de Máquina , Neoplasias da Próstata/patologia , Área Sob a Curva , Autoantígenos/genética , Autoantígenos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Imagem por Ressonância Magnética , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/genética , Curva ROC , Ribonuclease P/genética , Ribonuclease P/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
18.
Nat Commun ; 11(1): 1253, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152303

RESUMO

The presence of antiendothelial cell antibodies (AECAs) has been documented in Takayasu arteritis (TAK), a chronic granulomatous vasculitis. Here, we identify cell-surface autoantigens using an expression cloning system. A cDNA library of endothelial cells is retrovirally transfected into a rat myeloma cell line from which AECA-positive clones are sorted with flow cytometry. Four distinct AECA-positive clones are isolated, and endothelial protein C receptor (EPCR) and scavenger receptor class B type 1 (SR-BI) are identified as endothelial autoantigens. Autoantibodies against EPCR and SR-BI are detected in 34.6% and 36.5% of cases, respectively, with minimal overlap (3.8%). Autoantibodies against EPCR are also detected in ulcerative colitis, the frequent comorbidity of TAK. In mechanistic studies, EPCR and SR-BI function as negative regulators of endothelial activation. EPCR has also an effect on human T cells and impair Th17 differentiation. Autoantibodies against EPCR and SR-BI block the functions of their targets, thereby promoting pro-inflammatory phenotype.


Assuntos
Autoanticorpos/metabolismo , Autoantígenos/isolamento & purificação , Autoantígenos/metabolismo , Células Endoteliais/imunologia , Arterite de Takayasu/imunologia , Arterite de Takayasu/metabolismo , Animais , Autoanticorpos/isolamento & purificação , Autoantígenos/genética , Autoantígenos/imunologia , Linhagem Celular Tumoral , Membrana Celular/química , Clonagem Molecular , Colite Ulcerativa/imunologia , Modelos Animais de Doenças , Receptor de Proteína C Endotelial , Endotélio Vascular/metabolismo , Biblioteca Gênica , Humanos , Mieloma Múltiplo/metabolismo , Proteína C/metabolismo , Ratos , Receptores de Endotelina/metabolismo , Receptores Depuradores Classe B/metabolismo
19.
Nat Commun ; 11(1): 1386, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170061

RESUMO

Microglia maintain brain homeostasis by removing neuron-derived components such as myelin and cell debris. The evidence linking microglia to neurodegenerative diseases is growing; however, the precise mechanisms remain poorly understood. Herein, we report a neuroprotective role for microglia in the clearance of neuron-released α-synuclein. Neuronal α-synuclein activates microglia, which in turn engulf α-synuclein into autophagosomes for degradation via selective autophagy (termed synucleinphagy). Synucleinphagy requires the presence of microglial Toll-like receptor 4 (TLR4), which induces transcriptional upregulation of p62/SQSTM1 through the NF-κB signaling pathway. Induction of p62, an autophagy receptor, is necessary for the formation of α-synuclein/ubiquitin-positive puncta that are degraded by autophagy. Finally, disruption of microglial autophagy in mice expressing human α-synuclein promotes the accumulation of misfolded α-synuclein and causes midbrain dopaminergic neuron degeneration. Our study thus identifies a neuroprotective function of microglia in the clearance of α-synuclein via TLR4-NF-κB-p62 mediated synucleinphagy.


Assuntos
Autofagia/fisiologia , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptor 4 Toll-Like/metabolismo , alfa-Sinucleína/metabolismo , Animais , Autoantígenos/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Feminino , Células HEK293 , Humanos , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , NF-kappa B/metabolismo , Transdução de Sinais
20.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165714, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32023482

RESUMO

Blood-cell targeting Autoimmune Diseases (BLADs) are complex diseases that affect blood cell formation or prevent blood cell production. Since these clinical conditions are gathering growing attention, experimental approaches are being used to investigate the mechanisms behind their pathogenesis and to identify proteins associated with them. However, computational approaches have not been utilized extensively in the study of BLADs. This study aims to investigate the interaction network of proteins associated with BLADs (BLAD interactome) and to identify novel associations with other human proteins. The method followed in this study combines information regarding protein-protein interaction network properties and autoimmune disease terms. Proteins with high network scores and statistically significant autoimmune disease term enrichment were obtained and 14 of them were designated as candidate proteins associated with BLADs. Additionally, clustering analysis of the BLAD interactome was used and allowed the detection of 17 proteins that act as "connectors" of different BLADs. We expect our findings to further extend experimental efforts for the investigation of the pathogenesis and the relationships of BLADs.


Assuntos
Doenças Autoimunes/imunologia , Células Sanguíneas/imunologia , Doenças Hematológicas/imunologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/imunologia , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Autoantígenos/metabolismo , Doenças Autoimunes/sangue , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/metabolismo , Células Sanguíneas/metabolismo , Análise por Conglomerados , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Doenças Hematológicas/sangue , Hematopoese/imunologia , Humanos
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