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1.
Hum Mol Genet ; 25(8): 1600-18, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26908611

RESUMO

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.


Assuntos
Caspase 6/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina/genética , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinase 3 , Técnicas do Sistema de Duplo-Híbrido
2.
Genome Res ; 25(5): 701-13, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25908449

RESUMO

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.


Assuntos
Algoritmos , Proteínas do Tecido Nervoso/metabolismo , Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Drosophila/genética , Drosophila/metabolismo , Proteína Huntingtina , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Células PC12 , Ligação Proteica , Ratos
3.
Hum Mol Genet ; 23(15): 4142-60, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24705354

RESUMO

HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10(-5)). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.


Assuntos
Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Processamento de Proteína Pós-Traducional , Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Redes Reguladoras de Genes , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoilação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Técnicas do Sistema de Duplo-Híbrido
4.
Biogerontology ; 17(5-6): 817-828, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27465500

RESUMO

Death-associated protein 6 (DAXX) is a ubiquitous protein implicated in various cellular processes such as apoptosis, tumorigenesis, development and transcription. The role of DAXX is however ambiguous and many contradictory results regarding its function in apoptosis upon various cellular stresses are described in the literature. In order to have a better understanding of the role of DAXX throughout the entire organism under physiological stress conditions, we have characterized the mRNA levels, protein expression and the proteolytic processing of DAXX in the normal aging process in peripheral organs and brain regions in C57BL/6 male mice. Overall, Daxx mRNA expression decreases with aging in the liver, kidney, heart, cortex and cerebellum. In contrast, an increase is observed in the striatum. The protein expression of DAXX and of its proteolytic fragments increases with aging in the kidney, heart and cortex. In liver and spleen, no changes are observed while in the striatum and cerebellum, certain forms increase and others decrease with age, suggesting that the functions of DAXX may be cell type dependent. This study provides important details regarding the expression and post-translational modifications of DAXX in aging in the entire organism and provides reference data for the deregulation observed in age-associated diseases.


Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Vísceras/metabolismo , Animais , Proteínas Correpressoras , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Especificidade de Órgãos/fisiologia
5.
PLoS Genet ; 8(8): e1002897, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916034

RESUMO

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.


Assuntos
Complexo Mediador/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Peptídeos/química , Proteínas de Ligação a RNA/química , Animais , Ataxina-1 , Ataxinas , Células COS , Chlorocebus aethiops , Escherichia coli/genética , Humanos , Complexo Mediador/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Plasmídeos , Polimerização , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , Transfecção
6.
Mol Metab ; 60: 101468, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35248787

RESUMO

OBJECTIVES: Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis [ALS]), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We investigated intermediary metabolism of neurons expressing familial ALS (fALS) genes and interrogated the functional consequences of glycolysis genes in fitness assays and neuronal survival. METHODS: We created a pure neuronal model system for isotopologue investigations of fuel utilization. In a yeast platform we studied the functional contributions of glycolysis genes in a growth fitness assay iafter expressing of a fALS gene. RESULTS: We find in our rodent models of fALS, a reduction in neuronal lactate production with maintained or enhanced activity of the neuronal citric acid cycle. This rewiring of metabolism is associated with normal ATP levels, bioenergetics, and redox status, thus supporting the notion that gross mitochondrial function is not compromised in neurons soon after expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. CONCLUSIONS: We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated alteration in mitochondrial function in a baleful manner and targeting this process can be healthful.


Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Trifosfato de Adenosina , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
7.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33108356

RESUMO

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.


Assuntos
Envelhecimento/metabolismo , Microglia/enzimologia , Biossíntese de Proteínas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento/genética , Animais , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/genética , Serina-Treonina Quinases TOR/genética
8.
J Basic Microbiol ; 50(1): 83-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20175124

RESUMO

After dispiriting results using viral vectors in gene therapy, by which a number of patients acquired cancer as a result of the use of retroviral vector constructs, the percentage of non-viral approaches has increased over recent years. To elucidate potential bottlenecks in the non-viral transfection process we here introduce a novel method to directly visualize endocytic non-viral DNA uptake in a transfection approach. This novel method allows for the first time to monitor the location of DNA which is taken up by endocytosis in yeast (Saccharomyces cerevisiae) wild type and mutant strains. More specifically it enables drawing conclusions about conditions favouring non-viral gene transfection.


Assuntos
DNA/metabolismo , Endocitose , Saccharomyces cerevisiae/metabolismo , Transfecção/métodos , Sondas de DNA/química , Etídio/química , Fluoresceína/química , Corantes Fluorescentes/química , Vetores Genéticos/metabolismo , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética
9.
Cell Rep ; 32(7): 108050, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32814053

RESUMO

Interactome maps are valuable resources to elucidate protein function and disease mechanisms. Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. This network reveals interconnectivity across diseases and links many known ND-causing proteins, such as α-synuclein, TDP-43, and ATXN1, to a host of proteins previously unrelated to NDs. It facilitates the identification of interacting proteins that significantly influence mutant TDP-43 and HTT toxicity in transgenic flies, as well as of ARF-GEP100 that controls misfolding and aggregation of multiple ND-causing proteins in experimental model systems. Furthermore, it enables the prediction of ND-specific subnetworks and the identification of proteins, such as ATXN1 and MKL1, that are abnormally aggregated in postmortem brains of Alzheimer's disease patients, suggesting widespread protein aggregation in NDs.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiopatologia , Doenças Neurodegenerativas/genética , Agregados Proteicos/genética , Mapeamento de Interação de Proteínas/métodos , Humanos
10.
J Cell Biochem ; 106(2): 327-36, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19115284

RESUMO

The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non-viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH-value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the N+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non-viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole.


Assuntos
Ácidos , DNA Fúngico/metabolismo , Endocitose , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Transporte Biológico , Concentração de Íons de Hidrogênio , Mutação/genética , Saccharomyces cerevisiae/genética , Transgenes/genética
11.
Yeast ; 25(12): 871-7, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19160456

RESUMO

The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. A micro-scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter alpha-amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger-scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine alpha-amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here secretion and early endocytosis 1 protein (See1p), as a novel component of intracellular transport.


Assuntos
Endocitose , Deleção de Genes , Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Regulação da Expressão Gênica , Metiltransferases/genética , Camundongos , Poligalacturonase/genética , Poligalacturonase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transfecção , Proteínas de Transporte Vesicular/genética , alfa-Amilases/genética , alfa-Amilases/metabolismo
12.
Neurobiol Aging ; 47: 50-62, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27552481

RESUMO

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.


Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Apoptose/genética , Caspases/genética , Caspases/metabolismo , Expressão Gênica/genética , Especificidade de Órgãos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Animais , Encéfalo/metabolismo , Caspases/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/genética , Serina-Treonina Quinase 3
14.
Chembiochem ; 8(9): 1048-54, 2007 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-17471480

RESUMO

A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer.


Assuntos
Metiltransferases/química , Peptídeo Sintases/química , Catálise , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Epitopos , Escherichia coli/enzimologia , Fermentação , Fusarium/enzimologia , Cinética , Espectroscopia de Ressonância Magnética , Marcadores de Fotoafinidade , Proteínas Recombinantes/química , S-Adenosilmetionina , Saccharomyces cerevisiae/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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