RESUMEN
Autophagy ensures cellular homeostasis by the degradation of long-lived proteins, damaged organelles and pathogens. This catabolic process provides essential cellular building blocks upon nutrient deprivation. Cellular metabolism, especially mitochondrial respiration, has a significant influence on autophagic flux, and complex I function is required for maximal autophagy. In Parkinson's disease mitochondrial function is frequently impaired and autophagic flux is altered. Thus, dysfunctional organelles and protein aggregates accumulate and cause cellular damage. In order to investigate the interdependency between mitochondrial function and autophagy, novel tool compounds are required. Herein, we report the discovery of a structurally novel autophagy inhibitor (Authipyrin) using a high content screening approach. Target identification and validation led to the discovery that Authipyrin targets mitochondrial complex I directly, leading to the potent inhibition of mitochondrial respiration as well as autophagy.
Asunto(s)
Autofagia , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Mitocondrias/metabolismo , Pirina/química , Autofagia/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Humanos , Células MCF-7 , Proteínas Asociadas a Microtúbulos/metabolismo , Oligomicinas/farmacología , Pirina/metabolismo , Pirina/farmacologíaRESUMEN
Pyrin protein is the product of the MEFV gene, mutations in which cause manifestation of familial Mediterranean fever (FMF). Functions of pyrin are not completely clear. The secondary structure of the pyrin is represented with four domains and two motifs. Mutations p.M680I, p.M694V, p.M694I, p.K695R, p.V726A, and p.A744S, which are located in the B30.2 domain of pyrin protein, are responsible for manifestation of the most common and severe forms of FMF. All the domains and the motifs of pyrin, are directly or indirectly, involved in the protein-protein interaction with proteins of apoptosis and regulate the cascade of inflammatory reactions, which is impaired due to pyrin mutations. It is well known, that malfunction of the pyrin-caspase-1 complex is the main reason of inflammation during FMF. Complete tertiary structure of pyrin and the effects of mutations in it are experimentally not studied yet. The aim of this study was to identify possible effects of the abovementioned mutations in the B30.2 domain tertiary structure and to determine their potential consequences in formation of the B30.2-caspase-1 complex. Using in silico methods, it was found, that these mutations led to structural rearrangements in B30.2 domain tertiary structure, causing shifts of binding sites and altering the interaction energy between B30.2 and caspase-1.
Asunto(s)
Dominio B30.2-SPRY , Caspasa 1/química , Simulación de Dinámica Molecular , Pirina/química , Sitios de Unión , Fiebre Mediterránea Familiar , Humanos , Cinética , Mutación , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Targeted Next Generation Sequencing (NGS) assays are cost-efficient and reliable alternatives to Sanger sequencing. For sequencing of very large set of genes, the target enrichment approach is suitable. However, for smaller genomic regions, the target amplification method is more efficient than both the target enrichment method and Sanger sequencing. The major difficulty of the target amplification method is the preparation of amplicons, regarding required time, equipment, and labor. Multiplex PCR (MPCR) is a good solution for the mentioned problems. RESULTS: We propose a novel method to design MPCR primers for a continuous genomic region, following the best practices of clinically reliable PCR design processes. On an experimental setup with 48 different combinations of factors, we have shown that multiple parameters might effect finding the first feasible solution. Increasing the length of the initial primer candidate selection sequence gives better results whereas waiting for a longer time to find the first feasible solution does not have a significant impact. CONCLUSIONS: We generated MPCR primer designs for the HBB whole gene, MEFV coding regions, and human exons between 2000 bp to 2100 bp-long. Our benchmarking experiments show that the proposed MPCR approach is able produce reliable NGS assay primers for a given sequence in a reasonable amount of time.
Asunto(s)
Algoritmos , Cartilla de ADN/metabolismo , ADN/metabolismo , Reacción en Cadena de la Polimerasa Multiplex/métodos , ADN/química , Cartilla de ADN/química , Exones , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pirina/química , Pirina/genéticaRESUMEN
NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge-charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pirina/química , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Dominios Proteicos , Proteína Fosfatasa 2/metabolismo , Relación Estructura-ActividadRESUMEN
NLRP3 inflammasome is a multiprotein platform for the activation of caspase-1. Despite the increasing number of reports linking NLRP3 inflammasome to a variety of diseases, the mechanism behind the NLRP3 activation remains elusive, especially in terms of the early stages which are critical to the NLRP3 inflammasome assembly. In the present study we aimed to determine the minimal oligomerization state required for the NLRP3 inflammasome activation. For this purpose, NLRP3 pyrin domain (NLRP3PYD) was fused to various dimerization and trimerization domains. The constructs were expressed under the inducible promoter in mouse macrophages lacking endogenous NLRP3. Dimerization of the NLRP3PYD either in parallel or in antiparallel orientation was insufficient for the inflammasome activation. Trimerization of the NLRP3PYD with the foldon domain, however, induced pyroptosis and robust IL-1ß maturation, which was caspase-1 dependent. Interestingly, foldon-induced constitutive activation is resistant to inhibition with NLRP3-specific inhibitor MCC950 and does not lead to ASC speck formation. Although we cannot exclude that wild-type NLRP3 forms higher oligomer species similar to NLRP1 or NLRC4, our results clearly demonstrate that efficient IL-1ß response can be achieved by the induced trimerization of the NLRP3PYD domain.
Asunto(s)
Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Caspasa 1/metabolismo , Línea Celular , Activación Enzimática , Inflamasomas/inmunología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Dominios Proteicos , Multimerización de Proteína , Pirina/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.