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1.
Annu Rev Neurosci ; 41: 255-276, 2018 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-29661037

RESUMO

One of the fundamental properties of the cell is the capability to digest and remodel its own components according to metabolic and developmental needs. This is accomplished via the autophagy-lysosome system, a pathway of critical importance in the brain, where it contributes to neuronal plasticity and must protect nonreplaceable neurons from the potentially harmful accumulation of cellular waste. The study of lysosomal biogenesis and function in the context of common and rare neurodegenerative diseases has revealed that a dysfunctional autophagy-lysosome system is the shared nexus where multiple, interconnected pathogenic events take place. The characterization of pathways and mechanisms regulating the lysosomal system and autophagic clearance offers unprecedented opportunities for the development of polyvalent therapeutic strategies based on the enhancement of the autophagy-lysosome pathway to maintain cellular homeostasis and achieve neuroprotection.


Assuntos
Autofagia/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Lisossomos/fisiologia , Animais , Humanos , Metabolismo dos Lipídeos , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia
2.
Proc Natl Acad Sci U S A ; 116(8): 2977-2986, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30728291

RESUMO

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that causes tumor formation in multiple organs. TSC is caused by inactivating mutations in the genes encoding TSC1/2, negative regulators of the mammalian target of rapamycin complex 1 (mTORC1). Diminished TSC function is associated with excess glycogen storage, but the causative mechanism is unknown. By studying human and mouse cells with defective or absent TSC2, we show that complete loss of TSC2 causes an increase in glycogen synthesis through mTORC1 hyperactivation and subsequent inactivation of glycogen synthase kinase 3ß (GSK3ß), a negative regulator of glycogen synthesis. Specific TSC2 pathogenic mutations, however, result in elevated glycogen levels with no changes in mTORC1 or GSK3ß activities. We identify mTORC1-independent lysosomal depletion and impairment of autophagy as the driving causes underlying abnormal glycogen storage in TSC irrespective of the underlying mutation. The defective autophagic degradation of glycogen is associated with abnormal ubiquitination and degradation of essential proteins of the autophagy-lysosome pathway, such as LC3 and lysosomal associated membrane protein 1 and 2 (LAMP1/2) and is restored by the combined use of mTORC1 and Akt pharmacological inhibitors. In complementation to current models that place mTORC1 as the central therapeutic target for TSC pathogenesis, our findings identify mTORC1-independent pathways that are dysregulated in TSC and that should therefore be taken into account in the development of a therapeutic treatment.


Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Glicogênio/biossíntese , Proteína 2 do Complexo Esclerose Tuberosa/genética , Esclerose Tuberosa/genética , Animais , Autofagia/genética , Glicogênio/genética , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/genética , Lisossomos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mutação , Proteólise , Transdução de Sinais , Esclerose Tuberosa/patologia , Ubiquitinação/genética
3.
Am J Hum Genet ; 103(1): 154-162, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29961569

RESUMO

TRAF7 is a multi-functional protein involved in diverse signaling pathways and cellular processes. The phenotypic consequence of germline TRAF7 variants remains unclear. Here we report missense variants in TRAF7 in seven unrelated individuals referred for clinical exome sequencing. The seven individuals share substantial phenotypic overlap, with developmental delay, congenital heart defects, limb and digital anomalies, and dysmorphic features emerging as key unifying features. The identified variants are de novo in six individuals and comprise four distinct missense changes, including a c.1964G>A (p.Arg655Gln) variant that is recurrent in four individuals. These variants affect evolutionarily conserved amino acids and are located in key functional domains. Gene-specific mutation rate analysis showed that the occurrence of the de novo variants in TRAF7 (p = 2.6 × 10-3) and the recurrent de novo c.1964G>A (p.Arg655Gln) variant (p = 1.9 × 10-8) in our exome cohort was unlikely to have occurred by chance. In vitro analyses of the observed TRAF7 mutations showed reduced ERK1/2 phosphorylation. Our findings suggest that missense mutations in TRAF7 are associated with a multisystem disorder and provide evidence of a role for TRAF7 in human development.


Assuntos
Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Adulto , Aminoácidos/genética , Criança , Pré-Escolar , Exoma/genética , Feminino , Cardiopatias Congênitas/genética , Humanos , Lactente , Recém-Nascido , Sistema de Sinalização das MAP Quinases/genética , Masculino , Anormalidades Musculoesqueléticas/genética , Fenótipo
4.
PLoS Genet ; 13(6): e1006825, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28640802

RESUMO

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.


Assuntos
Metabolismo dos Carboidratos , Transtornos Peroxissômicos/genética , Peroxissomos/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glucose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Fator 2 da Biogênese de Peroxissomos , Peroxissomos/genética , Transcriptoma
5.
J Neurochem ; 148(5): 573-589, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30092616

RESUMO

This review focuses on the pathways that regulate lysosome biogenesis and that are implicated in numerous degenerative storage diseases, including lysosomal storage disorders and late-onset neurodegenerative diseases. Lysosomal proteins are synthesized in the endoplasmic reticulum and trafficked to the endolysosomal system through the secretory route. Several receptors have been characterized that execute post-Golgi trafficking of lysosomal proteins. Some of them recognize their cargo proteins based on specific amino acid signatures, others based on a particular glycan modification that is exclusively found on lysosomal proteins. Nearly all receptors serving lysosome biogenesis are under the transcriptional control of transcription factor EB (TFEB), a master regulator of the lysosomal system. TFEB coordinates the expression of lysosomal hydrolases, lysosomal membrane proteins, and autophagy proteins in response to pathways sensing lysosomal stress and the nutritional conditions of the cell among other stimuli. TFEB is primed for activation in lysosomal storage disorders but surprisingly its function is impaired in some late-onset neurodegenerative storage diseases like Alzheimer's and Parkinson's, because of specific detrimental interactions that limit TFEB expression or activation. Thus, disrupted TFEB function presumably plays a role in the pathogenesis of these diseases. Multiple studies in animal models of degenerative storage diseases have shown that exogenous expression of TFEB and pharmacological activation of endogenous TFEB attenuate disease phenotypes. These results highlight TFEB-mediated enhancement of lysosomal biogenesis and function as a candidate strategy to counteract the progression of these diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".


Assuntos
Doenças por Armazenamento dos Lisossomos , Lisossomos , Biogênese de Organelas , Animais , Humanos
6.
PLoS Biol ; 13(3): e1002103, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25811491

RESUMO

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.


Assuntos
Canais de Cálcio Tipo N/genética , Canais de Cálcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Fagossomos/metabolismo , Animais , Autofagia/genética , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio Tipo N/deficiência , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Proteínas de Drosophila/deficiência , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Endossomos/ultraestrutura , Feminino , Regulação da Expressão Gênica , Homeostase/genética , Lisossomos/ultraestrutura , Masculino , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/ultraestrutura , Fagossomos/ultraestrutura , Cultura Primária de Células , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura
7.
J Biol Chem ; 289(14): 10211-22, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24558044

RESUMO

2-Hydroxypropyl-ß-cyclodextrin (HPßCD) is a Food and Drug Administration-approved excipient used to improve the stability and bioavailability of drugs. Despite its wide use as a drug delivery vehicle and the recent approval of a clinical trial to evaluate its potential for the treatment of a cholesterol storage disorder, the cellular pathways involved in the adaptive response that is activated upon exposure to HPßCD are still poorly defined. Here, we show that cell treatment with HPßCD results in the activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and in enhancement of the cellular autophagic clearance capacity. HPßCD administration promotes transcription factor EB-mediated clearance of proteolipid aggregates that accumulate due to inefficient activity of the lysosome-autophagy system in cells derived from a patient with a lysosomal storage disorder. Interestingly, HPßCD-mediated activation of autophagy was found not to be associated with activation of apoptotic pathways. This study provides a mechanistic understanding of the cellular response to HPßCD treatment, which will inform the development of safe HPßCD-based therapeutic modalities and may enable engineering HPßCD as a platform technology to reduce the accumulation of lysosomal storage material.


Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Excipientes/farmacologia , Fibroblastos/metabolismo , Lisossomos/metabolismo , beta-Ciclodextrinas/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células HeLa , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/genética
8.
Hum Mol Genet ; 22(10): 1994-2009, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23393155

RESUMO

Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a ß-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Lisossomos/metabolismo , Dobramento de Proteína , Proteólise , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Fibroblastos , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Doença de Gaucher/terapia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Lisossomos/genética , Mutação , Transporte Proteico/fisiologia , Doença de Tay-Sachs/genética , Doença de Tay-Sachs/metabolismo , Doença de Tay-Sachs/terapia
9.
Genome Res ; 22(6): 1163-72, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22345618

RESUMO

MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFß pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFß signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs.


Assuntos
Redes Reguladoras de Genes , Genômica/métodos , MicroRNAs/genética , Genoma Humano , Humanos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo
10.
Nat Neurosci ; 27(1): 48-62, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37985800

RESUMO

Transcription factor EB (TFEB) mediates gene expression through binding to the coordinated lysosome expression and regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase), which are essential for lysosome acidification. Single-nucleus RNA sequencing of wild-type and PS19 (Tau) transgenic mice expressing the P301S mutant tau identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and were locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.


Assuntos
Tauopatias , ATPases Vacuolares Próton-Translocadoras , Animais , Camundongos , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Camundongos Transgênicos , Microglia/metabolismo , Transdução de Sinais/fisiologia , Tauopatias/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética
11.
Nat Metab ; 6(2): 359-377, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38409323

RESUMO

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.


Assuntos
Doenças Cardiovasculares , Humanos , Masculino , Feminino , Camundongos , Animais , Leucina/metabolismo , Leucina/farmacologia , Fatores de Risco , Serina-Treonina Quinases TOR/metabolismo , Macrófagos/metabolismo , Fatores de Risco de Doenças Cardíacas , Mamíferos/metabolismo
12.
Hum Mol Genet ; 20(19): 3852-66, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21752829

RESUMO

In metazoans, lysosomes are the center for the degradation of macromolecules and play a key role in a variety of cellular processes, such as autophagy, exocytosis and membrane repair. Defects of lysosomal pathways are associated with lysosomal storage disorders and with several late onset neurodegenerative diseases. We recently discovered the CLEAR (Coordinated Lysosomal Expression and Regulation) gene network and its master gene transcription factor EB (TFEB), which regulates lysosomal biogenesis and function. Here, we used a combination of genomic approaches, including ChIP-seq (sequencing of chromatin immunoprecipitate) analysis, profiling of TFEB-mediated transcriptional induction, genome-wide mapping of TFEB target sites and recursive expression meta-analysis of TFEB targets, to identify 471 TFEB direct targets that represent essential components of the CLEAR network. This analysis revealed a comprehensive system regulating the expression, import and activity of lysosomal enzymes that control the degradation of proteins, glycosaminoglycans, sphingolipids and glycogen. Interestingly, the CLEAR network appears to be involved in the regulation of additional lysosome-associated processes, including autophagy, exo- and endocytosis, phagocytosis and immune response. Furthermore, non-lysosomal enzymes involved in the degradation of essential proteins such as hemoglobin and chitin are also part of the CLEAR network. Finally, we identified nine novel lysosomal proteins by using the CLEAR network as a tool for prioritizing candidates. This study provides potential therapeutic targets to modulate cellular clearance in a variety of disease conditions.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Lisossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células HeLa , Humanos , Lisossomos/enzimologia , Lisossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo
13.
bioRxiv ; 2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36798205

RESUMO

Transcription factor EB (TFEB) mediates gene expression through binding to the Coordinated Lysosome Expression And Regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase) essential for lysosome acidification. Single nucleus RNA-sequencing (snRNA-seq) of wild-type and PS19 (Tau) transgenic mice identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. We show that the CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and was locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homoeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.

14.
Cell Discov ; 9(1): 32, 2023 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-36964131

RESUMO

Calpains are a class of non-lysosomal cysteine proteases that exert their regulatory functions via limited proteolysis of their substrates. Similar to the lysosomal and proteasomal systems, calpain dysregulation is implicated in the pathogenesis of neurodegenerative disease and cancer. Despite intensive efforts placed on the identification of mechanisms that regulate calpains, however, calpain protein modifications that regulate calpain activity are incompletely understood. Here we show that calpains are regulated by KCTD7, a cytosolic protein of previously uncharacterized function whose pathogenic mutations result in epilepsy, progressive ataxia, and severe neurocognitive deterioration. We show that KCTD7 works in complex with Cullin-3 and Rbx1 to execute atypical, non-degradative ubiquitination of calpains at specific sites (K398 of calpain 1, and K280 and K674 of calpain 2). Experiments based on single-lysine mutants of ubiquitin determined that KCTD7 mediates ubiquitination of calpain 1 via K6-, K27-, K29-, and K63-linked chains, whereas it uses K6-mediated ubiquitination to modify calpain 2. Loss of KCTD7-mediated ubiquitination of calpains led to calpain hyperactivation, aberrant cleavage of downstream targets, and caspase-3 activation. CRISPR/Cas9-mediated knockout of Kctd7 in mice phenotypically recapitulated human KCTD7 deficiency and resulted in calpain hyperactivation, behavioral impairments, and neurodegeneration. These phenotypes were largely prevented by pharmacological inhibition of calpains, thus demonstrating a major role of calpain dysregulation in KCTD7-associated disease. Finally, we determined that Cullin-3-KCTD7 mediates ubiquitination of all ubiquitous calpains. These results unveil a novel mechanism and potential target to restrain calpain activity in human disease and shed light on the molecular pathogenesis of KCTD7-associated disease.

15.
Nucleic Acids Res ; 36(Database issue): D741-6, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17942430

RESUMO

We have developed a comprehensive resource devoted to biologists wanting to optimize the use of gene trap clones in their experiments. We have processed 300 602 such clones from both public and private projects to generate 28,199 'UniTraps', i.e. distinct collections of unambiguous insertions at the same subgenic region of annotated genes. The UniTrap resource contains data relative to 9583 trapped genes, which represent 42.3% of the mouse gene content. Among the trapped genes, 7,728 have a counterpart in humans, and 677 are known to be involved in the pathogenesis of human diseases. The aim of this analysis is to provide the wet lab researchers with a comprehensive database and curated tools for (i) identifying and comparing the clones carrying a trap into the genes of interest, (ii) evaluating the severity of the mutation to the protein function in each independent trapping event and (iii) supplying complete information to perform PCR, RT-PCR and restriction experiments to verify the clone and identify the exact point of vector insertion. To share this unique resource with the scientific community, we have designed and implemented a web interface that is freely accessible at http://unitrap.cbm.fvg.it/.


Assuntos
Bases de Dados Genéticas , Camundongos/genética , Mutagênese Insercional , Animais , Mapeamento Cromossômico , Células Clonais , Vetores Genéticos , Humanos , Internet , Reação em Cadeia da Polimerase , Proteínas/genética , Proteínas/fisiologia , Software , Interface Usuário-Computador
16.
Nucleic Acids Res ; 36(13): 4402-16, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18596081

RESUMO

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5'RACE and 3'RACE we identified three IBTKalpha, IBTKbeta and IBTKgamma mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk.


Assuntos
Proteínas de Transporte/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Linhagem Celular , Biologia Computacional , Evolução Molecular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Regiões Promotoras Genéticas , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/química
17.
J Clin Invest ; 130(8): 4118-4132, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32597833

RESUMO

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.


Assuntos
Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Lisossomos/enzimologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Lisossomos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Transporte Proteico/genética
18.
Autophagy ; 15(7): 1214-1233, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30741620

RESUMO

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. We find that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. We found that loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. We propose that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation. Abbreviations: ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; CERT: ceramide transfer protein; FFAT: two phenylalanines in an acidic tract; MSP: major sperm proteins; OSBP: oxysterol binding protein; PH: pleckstrin homology; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns4K: phosphatidylinositol 4-kinase; UPR: unfolded protein response; VAMP: vesicle-associated membrane protein; VAPA/B: mammalian VAPA and VAPB proteins; VAPs: VAMP-associated proteins (referring to Drosophila Vap33, and human VAPA and VAPB).


Assuntos
Autofagia/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/efeitos dos fármacos , Endossomos/genética , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas R-SNARE/genética , eIF-2 Quinase/química , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
19.
BMC Evol Biol ; 8: 225, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18673550

RESUMO

BACKGROUND: The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection. RESULTS: Here we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures. CONCLUSION: We found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.


Assuntos
Evolução Molecular , Genoma Humano/genética , Família Multigênica , Isoformas de Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Processamento Alternativo , Motivos de Aminoácidos/genética , Animais , Biologia Computacional , Genômica , Humanos , Filogenia , Domínios RING Finger , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
20.
Trends Genet ; 21(1): 12-6, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15680507

RESUMO

In metazoan organisms, energy production is the only example of a process that is under dual genetic control: nuclear and mitochondrial. We used a genomic approach to examine how energy genes of both the nuclear and mitochondrial genomes are coordinated, and discovered a novel genetic regulatory circuit in Drosophila melanogaster that is surprisingly simple and parsimonious. This circuit is based on a single DNA regulatory element and can explain both intra- and inter-genomic coordinated expression of genes involved in energy production, including the full complement of mitochondrial and nuclear oxidative phosphorylation genes, and the genes involved in the Krebs cycle.


Assuntos
Drosophila melanogaster/genética , Metabolismo Energético/genética , Genoma , Animais , Anopheles/genética , DNA Mitocondrial/genética , Drosophila/genética
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