RESUMO
BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.
Assuntos
Cinesinas , Osteogênese Imperfeita , Animais , Humanos , Camundongos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Regulação para Baixo , Cinesinas/genética , Cinesinas/metabolismo , Células NIH 3T3 , Proteômica , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.
Assuntos
Deficiência Intelectual , Megalencefalia , Transtornos do Neurodesenvolvimento , Animais , Humanos , Criança , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Caenorhabditis elegans/metabolismo , Transtornos do Neurodesenvolvimento/genética , Deficiência Intelectual/genética , Fenótipo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Megalencefalia/genética , Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.
RESUMO
Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.
Assuntos
Variação Genética/genética , Precursores de Proteínas/genética , Proteína C Associada a Surfactante Pulmonar/genética , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas rab5 de Ligação ao GTP/genética , Células Epiteliais Alveolares/metabolismo , Animais , Caenorhabditis elegans/genética , Humanos , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/genética , Surfactantes Pulmonares/metabolismoRESUMO
BACKGROUND & AIMS: Insulin signaling is known to regulate essential proteostasis mechanisms. METHODS: The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. RESULTS: We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator-activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. CONCLUSIONS: These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies.
Assuntos
Insulina , Fígado , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Deficiência de alfa 1-Antitripsina , Animais , Insulina/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Mamíferos/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologiaRESUMO
We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI = 1, o/e = 0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the proband's phenotypes.
Assuntos
Deficiências do Desenvolvimento , Proteínas F-Box , Microcefalia , Proteínas Nucleares , Fosfoproteínas Fosfatases , Convulsões , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Criança , Deficiências do Desenvolvimento/genética , Proteínas F-Box/genética , Humanos , Microcefalia/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Convulsões/genética , Separase/genéticaRESUMO
Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.
Assuntos
Autofagia/genética , Interferon gama/toxicidade , Células Mieloides/patologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citoproteção/efeitos dos fármacos , Genoma , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/ultraestrutura , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Neurobeachin (NBEA) was initially identified as a candidate gene for autism. Recently, variants in NBEA have been associated with neurodevelopmental delay and childhood epilepsy. Here, we report on a novel NBEA missense variant (c.5899G > A, p.Gly1967Arg) in the Domain of Unknown Function 1088 (DUF1088) identified in a child enrolled in the Undiagnosed Diseases Network (UDN), who presented with neurodevelopmental delay and seizures. Modeling of this variant in the Caenorhabditis elegans NBEA ortholog, sel-2, indicated that the variant was damaging to in vivo function as evidenced by altered cell fate determination and trafficking of potassium channels in neurons. The variant effect was indistinguishable from that of the reference null mutation suggesting that the variant is a strong hypomorph or a complete loss-of-function. Our experimental data provide strong support for the molecular diagnosis and pathogenicity of the NBEA p.Gly1967Arg variant and the importance of the DUF1088 for NBEA function.
Assuntos
Proteínas de Transporte/genética , Epilepsia/genética , Variação Genética , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Criança , Feminino , Edição de Genes , Humanos , Patologia Molecular , Canais de Potássio/metabolismoRESUMO
ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase-mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild-type (WT-ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body-like vesicle diameter and intracellular ABCA3 trafficking using fluorescence-based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well-organized lamellar body-like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well-organized lamellar body-like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT-ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Células A549 , Vesículas Citoplasmáticas , Humanos , Doenças Pulmonares Intersticiais/genética , Mutação de Sentido Incorreto , Alvéolos Pulmonares , Surfactantes PulmonaresRESUMO
Autophagy plays an essential role in cell survival/death and functioning. Modulation of autophagy has been recognized as a promising therapeutic strategy against diseases/disorders associated with uncontrolled growth or accumulation of biomolecular aggregates, organelles, or cells including those caused by cancer, aging, neurodegeneration, and liver diseases such as α1-antitrypsin deficiency. Numerous pharmacological agents that enhance or suppress autophagy have been discovered. However, their molecular mechanisms of action are far from clear. Here, we collected a set of 225 autophagy modulators and carried out a comprehensive quantitative systems pharmacology (QSP) analysis of their targets using both existing databases and predictions made by our machine learning algorithm. Autophagy modulators include several highly promiscuous drugs (e.g., artenimol and olanzapine acting as activators, fostamatinib as an inhibitor, or melatonin as a dual-modulator) as well as selected drugs that uniquely target specific proteins (~30% of modulators). They are mediated by three layers of regulation: (i) pathways involving core autophagy-related (ATG) proteins such as mTOR, AKT, and AMPK; (ii) upstream signaling events that regulate the activity of ATG pathways such as calcium-, cAMP-, and MAPK-signaling pathways; and (iii) transcription factors regulating the expression of ATG proteins such as TFEB, TFE3, HIF-1, FoxO, and NF-κB. Our results suggest that PKA serves as a linker, bridging various signal transduction events and autophagy. These new insights contribute to a better assessment of the mechanism of action of autophagy modulators as well as their side effects, development of novel polypharmacological strategies, and identification of drug repurposing opportunities.
Assuntos
Autofagia/efeitos dos fármacos , Descoberta de Drogas/métodos , Farmacologia/métodos , Autofagia/genética , Biomarcadores , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Biologia Computacional/métodos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Pretreatment serum squamous cell carcinoma antigen (SCCA) is a prognostic biomarker in women with cervical cancer. SCCA has not been evaluated as an early indicator of response to chemoradiation therapy (CRT). The molecular role of the two SCCA isoforms, SCCA1 (SERPINB3) and SCCA2 (SERPINB4), in cervical cancer is unknown. We hypothesised that changes in serum SCCA during definitive CRT predicts treatment response, and that SCCA1 mediates radiation resistance. METHODS: Patients treated with definitive CRT for cervical squamous carcinoma with serum SCCA measured were included. SCCA immunohistochemistry was performed on tumour biopsies. Post-treatment FDG-PET/CT, recurrence, and overall survival were recorded. Radiation response of cervical tumour cell lines after SCCA1 expression or CRISPR/Cas9 knockout was evaluated by clonogenic survival assay. RESULTS: Persistently elevated serum SCCA during definitive CRT was an independent predictor of positive post-therapy FDG-PET/CT (P=0.043), recurrence (P=0.0046) and death (P=0.015). An SCCA1-expressing vector increased radioresistance, while SCCA knock out increased radiosensitivity of cervical tumour cell lines in vitro. CONCLUSIONS: Early response assessment with serum SCCA is a powerful prognostic tool. These findings suggest that escalation of therapy in patients with elevated or sustained serum SCCA and molecular targeting of SCCA1 should be considered.
Assuntos
Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/terapia , Quimiorradioterapia/métodos , Serpinas/sangue , Serpinas/metabolismo , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Fracionamento da Dose de Radiação , Feminino , Técnicas de Silenciamento de Genes , Humanos , Pessoa de Meia-Idade , Serpinas/genética , Análise de Sobrevida , Resultado do Tratamento , Regulação para Cima , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/metabolismoRESUMO
Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC-4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain, and extracellular domain) and sites (residues 130-132 and 134-137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel's responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel's response to mechanical forces.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/metabolismo , Estresse Mecânico , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , XenopusRESUMO
Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.
Assuntos
Autofagia/efeitos dos fármacos , Terapia Genética , Deficiência de alfa 1-Antitripsina/terapia , Animais , Autofagia/genética , Modelos Animais de Doenças , Células Epiteliais/patologia , Pulmão/patologia , Camundongos , Deficiência de alfa 1-Antitripsina/tratamento farmacológico , Deficiência de alfa 1-Antitripsina/patologiaRESUMO
α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options.
Assuntos
Descoberta de Drogas , Estudo de Associação Genômica Ampla , Interferência de RNA , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Animais , Caenorhabditis elegans , Biologia Computacional , Modelos Animais de Doenças , Genômica , Ensaios de Triagem em Larga Escala , Humanos , Mutação , Ligação Proteica , Deficiências na Proteostase/genética , Reprodutibilidade dos Testes , Deficiência de alfa 1-Antitripsina/tratamento farmacológicoRESUMO
The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Interferência de RNA , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Degradação Associada com o Retículo Endoplasmático , Expressão Gênica , Genes Reporter , Humanos , Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Regiões Promotoras Genéticas , Proteólise , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serpinas , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases.
Assuntos
Granzimas/antagonistas & inibidores , Serina Endopeptidases/efeitos dos fármacos , Serpinas/metabolismo , Granzimas/metabolismo , Humanos , Cinética , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Caenorhabditis elegans is a useful model organism for combining multiple imaging, genetic, and biochemical methodologies to gain more insight into the biological function of specific proteins. Combining both biochemical and genetic analyses can lead to a better understanding of how a given protein may function within the context of a network of other proteins or specific pathway. Here, we describe a protocol for the biochemical isolation of serpin-interacting proteins using affinity purification and proteomic analysis. As the knowledge of in vivo serpin interacting partners in C. elegans has largely been obtained using genetic and in vitro recombinant protein studies, this protocol serves as a complementary approach to provide insight into the biological function and regulation of serpins.
Assuntos
Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteômica/métodos , Proteínas Recombinantes/metabolismo , Serpinas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia de Afinidade , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteínas Recombinantes/isolamento & purificação , Serpinas/químicaRESUMO
The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease.
Assuntos
Serpinas/genética , Serpinas/fisiologia , Animais , Modelos Animais de Doenças , Evolução Molecular , Variação Genética , Humanos , Camundongos , Família Multigênica , Filogenia , Conformação ProteicaRESUMO
Aging is a common risk factor in neurodegenerative disorders. Investigating neuronal aging in an isogenic background stands to facilitate analysis of the interplay between neuronal aging and neurodegeneration. Here we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs) in Huntington's disease identified pathways involving RCAN1, a negative regulator of calcineurin. Notably, RCAN1 protein increased with age in reprogrammed MSNs as well as in human postmortem striatum and RCAN1 knockdown rescued patient-derived MSNs of Huntington's disease from degeneration. RCAN1 knockdown enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, leading to TFEB's nuclear localization by dephosphorylation. Furthermore, G2-115, an analog of glibenclamide with autophagy-enhancing activities, reduced the RCAN1-calcineurin interaction, phenocopying the effect of RCAN1 knockdown. Our results demonstrate that targeting RCAN1 genetically or pharmacologically can increase neuronal resilience in Huntington's disease.