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1.
Proc Natl Acad Sci U S A ; 119(49): e2212533119, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36442106

RESUMO

Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, is not well characterized. In this study, we compared the expression level of secreted proteases, lipases, phospholipases, and sphingomyelinases of Malassezia globosa in healthy subjects and seborrheic dermatitis or atopic dermatitis patients. We observed upregulated gene expression of the previously characterized secretory aspartyl protease MGSAP1 in both diseased groups, in lesional and non-lesional skin sites, as compared to healthy subjects. To explore the functional roles of MGSAP1 in skin disease, we generated a knockout mutant of the homologous protease MFSAP1 in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, further highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.


Assuntos
Ácido Aspártico Proteases , Dermatite Atópica , Malassezia , Humanos , Animais , Camundongos , Peptídeo Hidrolases/genética , Malassezia/genética , Inflamação , Ácido Aspártico Endopeptidases
2.
J Biol Chem ; 293(49): 18864-18878, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30291141

RESUMO

The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1ß. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Ácidos Borônicos/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mutação em Linhagem Germinativa , Células HEK293 , Humanos , Inflamação/genética , Mutação de Sentido Incorreto , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos
3.
EMBO Mol Med ; 15(5): e17078, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-37066513

RESUMO

Somatic and germline gain-of-function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor-prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss-of-function of the proto-oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress-induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.


Assuntos
Síndrome de Noonan , Receptores Proteína Tirosina Quinases , Humanos , Recém-Nascido , Desenvolvimento Embrionário/genética , Coração , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Xenopus laevis/genética
4.
Nat Cell Biol ; 25(5): 765-777, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37095322

RESUMO

PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Neoplasias Renais/metabolismo , NF-kappa B/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética
5.
Science ; 377(6603): 328-335, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35857590

RESUMO

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.


Assuntos
Inflamassomos , MAP Quinase Quinase Quinases , Proteínas NLR , Piroptose , Ribossomos , Estresse Fisiológico , Anisomicina/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamassomos/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , MAP Quinase Quinase Quinases/metabolismo , Mutação , Proteínas NLR/genética , Proteínas NLR/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Piroptose/efeitos dos fármacos , Piroptose/efeitos da radiação , Ribossomos/efeitos dos fármacos , Ribossomos/efeitos da radiação , Raios Ultravioleta
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