RESUMO
In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.
Assuntos
Dobramento de Proteína , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Inibidores de Proteassoma/farmacologia , Resposta ao Choque Térmico , Bortezomib/farmacologiaRESUMO
Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine-beta-synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.
Assuntos
Substituição de Aminoácidos , Biologia Computacional/métodos , Cistationina beta-Sintase/genética , Cistationina/metabolismo , Cistationina beta-Sintase/metabolismo , Homocisteína/metabolismo , Humanos , Fenótipo , Medicina de PrecisãoRESUMO
Emerging evidence suggested a converging mechanism in neurodegenerative brain diseases (NBD) involving early neuronal network dysfunctions and alterations in the homeostasis of neuronal firing as culprits of neurodegeneration. In this study, we used paired-end short-read and direct long-read whole genome sequencing to investigate an unresolved autosomal dominant dementia family significantly linked to 7q36. We identified and validated a chromosomal inversion of ca. 4 Mb, segregating on the disease haplotype and disrupting the coding sequence of dipeptidyl-peptidase 6 gene (DPP6). DPP6 resequencing identified significantly more rare variants-nonsense, frameshift, and missense-in early-onset Alzheimer's disease (EOAD, p value = 0.03, OR = 2.21 95% CI 1.05-4.82) and frontotemporal dementia (FTD, p = 0.006, OR = 2.59, 95% CI 1.28-5.49) patient cohorts. DPP6 is a type II transmembrane protein with a highly structured extracellular domain and is mainly expressed in brain, where it binds to the potassium channel Kv4.2 enhancing its expression, regulating its gating properties and controlling the dendritic excitability of hippocampal neurons. Using in vitro modeling, we showed that the missense variants found in patients destabilize DPP6 and reduce its membrane expression (p < 0.001 and p < 0.0001) leading to a loss of protein. Reduced DPP6 and/or Kv4.2 expression was also detected in brain tissue of missense variant carriers. Loss of DPP6 is known to cause neuronal hyperexcitability and behavioral alterations in Dpp6-KO mice. Taken together, the results of our genomic, genetic, expression and modeling analyses, provided direct evidence supporting the involvement of DPP6 loss in dementia. We propose that loss of function variants have a higher penetrance and disease impact, whereas the missense variants have a variable risk contribution to disease that can vary from high to low penetrance. Our findings of DPP6, as novel gene in dementia, strengthen the involvement of neuronal hyperexcitability and alteration in the homeostasis of neuronal firing as a disease mechanism to further investigate.
Assuntos
Inversão Cromossômica , Demência/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Mutação , Proteínas do Tecido Nervoso/deficiência , Doenças Neurodegenerativas/genética , Neurônios/fisiologia , Canais de Potássio/deficiência , Potenciais de Ação/fisiologia , Adulto , Idoso , Cromossomos Humanos Par 7/genética , Demência/fisiopatologia , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/fisiologia , Feminino , Genes Dominantes , Homeostase , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Linhagem , Penetrância , Polimorfismo de Nucleotídeo Único , Canais de Potássio/genética , Canais de Potássio/fisiologia , Estabilidade Proteica , Transporte Proteico , Transmissão Sináptica , Sequenciamento Completo do GenomaRESUMO
The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Engenharia de Proteínas , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Superóxido Dismutase-1/químicaRESUMO
Over 40% of proteins in any eukaryotic genome encode intrinsically disordered regions (IDRs) that do not adopt defined tertiary structures. Certain IDRs perform critical functions, but discovering them is non-trivial as the biological context determines their function. We present IDR-Screen, a framework to discover functional IDRs in a high-throughput manner by simultaneously assaying large numbers of DNA sequences that code for short disordered sequences. Functionality-conferring patterns in their protein sequence are inferred through statistical learning. Using yeast HSF1 transcription factor-based assay, we discovered IDRs that function as transactivation domains (TADs) by screening a random sequence library and a designed library consisting of variants of 13 diverse TADs. Using machine learning, we find that segments devoid of positively charged residues but with redundant short sequence patterns of negatively charged and aromatic residues are a generic feature for TAD functionality. We anticipate that investigating defined sequence libraries using IDR-Screen for specific functions can facilitate discovering novel and functional regions of the disordered proteome as well as understand the impact of natural and disease variants in disordered segments.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Ativação Transcricional , Clonagem Molecular , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Aprendizado de Máquina , Proteoma/genética , Análise de Sequência de DNARESUMO
Soluble functional proteins may transform into insoluble amyloid fibrils that deposit in a variety of tissues. Amyloid formation is a hallmark of age-related degenerative disorders. Perhaps surprisingly, amyloid fibrils can also be beneficial and are frequently exploited for diverse functional roles in organisms. Here we introduce AmyPro, an open-access database providing a comprehensive, carefully curated collection of validated amyloid fibril-forming proteins from all kingdoms of life classified into broad functional categories (http://amypro.net). In particular, AmyPro provides the boundaries of experimentally validated amyloidogenic sequence regions, short descriptions of the functional relevance of the proteins and their amyloid state, a list of the experimental techniques applied to study the amyloid state, important structural/functional/variation/mutation data transferred from UniProt, a list of relevant PDB structures categorized according to protein states, database cross-references and literature references. AmyPro greatly improves on similar currently available resources by incorporating both prions and functional amyloids in addition to pathogenic amyloids, and allows users to screen their sequences against the entire collection of validated amyloidogenic sequence fragments. By enabling further elucidation of the sequential determinants of amyloid fibril formation, we hope AmyPro will enhance the development of new methods for the precise prediction of amyloidogenic regions within proteins.
Assuntos
Proteínas Amiloidogênicas/química , Bases de Dados de Proteínas , Interface Usuário-ComputadorRESUMO
Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neoplasias do Colo/genética , Glioblastoma/genética , Corpos de Inclusão Intranuclear/metabolismo , Agregação Patológica de Proteínas/genética , Deficiências na Proteostase/genética , Proteína Supressora de Tumor p53/genética , Biópsia , Linhagem Celular Tumoral , Neoplasias do Colo/complicações , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citoplasma/metabolismo , Glioblastoma/complicações , Glioblastoma/metabolismo , Glioblastoma/patologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Humanos , Estimativa de Kaplan-Meier , Mutação , Agregação Patológica de Proteínas/etiologia , Agregação Patológica de Proteínas/metabolismo , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/metabolismo , Receptores sigma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Protein aggregation is a major factor limiting the biotechnological and therapeutic application of many proteins, including enzymes and monoclonal antibodies. The molecular principles underlying aggregation are by now sufficiently understood to allow rational redesign of natural polypeptide sequences for decreased aggregation tendency, and hence potentially increased expression and solubility. Given that aggregation-prone regions (APRs) tend to contribute to the stability of the hydrophobic core or to functional sites of the protein, mutations in these regions have to be carefully selected in order not to disrupt protein structure or function. Therefore, we here provide access to an automated pipeline to identify mutations that reduce protein aggregation by reducing the intrinsic aggregation propensity of the sequence (using the TANGO algorithm), while taking care not to disrupt the thermodynamic stability of the native structure (using the empirical force-field FoldX). Moreover, by providing a plot of the intrinsic aggregation propensity score of APRs corrected by the local stability of that region in the folded structure, we allow users to prioritize those regions in the protein that are most in need of improvement through protein engineering. The method can be accessed at http://solubis.switchlab.org/.
Assuntos
Biologia Computacional/métodos , Internet , Mutação , Agregados Proteicos/genética , Proteínas/química , Proteínas/genética , Software , Algoritmos , Bases de Dados de Proteínas , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Termodinâmica , Interface Usuário-ComputadorRESUMO
Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica/genética , Núcleo Celular/metabolismo , Dipeptídeos/química , Drosophila melanogaster/genética , Demência Frontotemporal/genética , Genes de Insetos , Testes Genéticos , Sequências Repetitivas de Aminoácidos , Transporte Ativo do Núcleo Celular/genética , Animais , Arginina/metabolismo , Modelos Animais de Doenças , Olho/patologia , Células HeLa , Humanos , Metilação , Interferência de RNARESUMO
Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.
Assuntos
Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Western Blotting , Linhagem Celular Tumoral , Cromatografia em Gel , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Mutação , Estabilidade Proteica , Solubilidade , alfa-Galactosidase/química , alfa-Galactosidase/genéticaRESUMO
OBJECTIVE: To assess the genetic contribution of TBK1, a gene implicated in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and FTD-ALS, in Belgian FTD and ALS patient cohorts containing a significant part of genetically unresolved patients. METHODS: We sequenced TBK1 in a hospital-based cohort of 482 unrelated patients with FTD and FTD-ALS and 147 patients with ALS and an extended Belgian FTD-ALS family DR158. We followed up mutation carriers by segregation studies, transcript and protein expression analysis, and immunohistochemistry. RESULTS: We identified 11 patients carrying a loss-of-function (LOF) mutation resulting in an overall mutation frequency of 1.7% (11/629), 1.1% in patients with FTD (5/460), 3.4% in patients with ALS (5/147), and 4.5% in patients with FTD-ALS (1/22). We found 1 LOF mutation, p.Glu643del, in 6 unrelated patients segregating with disease in family DR158. Of 2 mutation carriers, brain and spinal cord was characterized by TDP-43-positive pathology. The LOF mutations including the p.Glu643del mutation led to loss of transcript or protein in blood and brain. CONCLUSIONS: TBK1 LOF mutations are the third most frequent cause of clinical FTD in the Belgian clinically based patient cohort, after C9orf72 and GRN, and the second most common cause of clinical ALS after C9orf72. These findings reinforce that FTD and ALS belong to the same disease continuum.
Assuntos
Demência Frontotemporal/diagnóstico , Demência Frontotemporal/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bélgica/epidemiologia , Estudos de Coortes , Feminino , Demência Frontotemporal/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Protein aggregation is a hallmark of over 30 human pathologies. In these diseases, the aggregation of one or a few specific proteins is often toxic, leading to cellular degeneration and/or organ disruption in addition to the loss-of-function resulting from protein misfolding. Although the pathophysiological consequences of these diseases are overt, the molecular dysregulations leading to aggregate toxicity are still unclear and appear to be diverse and multifactorial. The molecular mechanisms of protein aggregation and therefore the biophysical parameters favoring protein aggregation are better understood. Here we perform an in silico survey of the impact of human sequence variation on the aggregation propensity of human proteins. We find that disease-associated variations are statistically significantly enriched in mutations that increase the aggregation potential of human proteins when compared to neutral sequence variations. These findings suggest that protein aggregation might have a broader impact on human disease than generally assumed and that beyond loss-of-function, the aggregation of mutant proteins involved in cancer, immune disorders or inflammation could potentially further contribute to disease by additional burden on cellular protein homeostasis.
Assuntos
Predisposição Genética para Doença/genética , Mutação/genética , Polimorfismo Genético/genética , Agregação Patológica de Proteínas/genética , Biologia Computacional , Bases de Dados Factuais , Humanos , Estabilidade ProteicaRESUMO
MOTIVATION: Protein aggregation is associated with a number of protein misfolding diseases and is a major concern for therapeutic proteins. Aggregation is caused by the presence of aggregation-prone regions (APRs) in the amino acid sequence of the protein. The lower the aggregation propensity of APRs and the better they are protected by native interactions within the folded structure of the protein, the more aggregation is prevented. Therefore, both the local thermodynamic stability of APRs in the native structure and their intrinsic aggregation propensity are a key parameter that needs to be optimized to prevent protein aggregation. RESULTS: The Solubis method presented here automates the process of carefully selecting point mutations that minimize the intrinsic aggregation propensity while improving local protein stability.
Assuntos
Mutação/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Análise de Sequência de Proteína/métodos , Software , Bases de Dados de Proteínas , Humanos , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas/metabolismo , TermodinâmicaRESUMO
Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 µm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.
Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Peptídeos/metabolismo , Agregados Proteicos , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Sequência de Aminoácidos , Citocalasina D/farmacologia , Proteínas de Ligação a DNA/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Lovastatina/farmacologia , Lisossomos/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteólise , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein ß-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichia coli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.
Assuntos
Proteínas de Bactérias/genética , Agregados Proteicos , Mapas de Interação de Proteínas , beta-Galactosidase/genética , Proteínas de Bactérias/metabolismo , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Antígeno Prostático Específico/sangue , Antígeno Prostático Específico/genética , Ligação Proteica/genética , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , beta-Galactosidase/metabolismoRESUMO
Owing to its association with a diverse range of human diseases, the determinants of protein aggregation are studied intensively. It is generally accepted that the effective aggregation tendency of a protein depends on many factors such as folding efficiency towards the native state, thermodynamic stability of that conformation, intrinsic aggregation propensity of the polypeptide sequence and its ability to be recognized by the protein quality control system. The intrinsic aggregation propensity of a polypeptide sequence is related to the presence of short APRs (aggregation-prone regions) that self-associate to form intermolecular ß-structured assemblies. These are typically short sequence segments (5-15 amino acids) that display high hydrophobicity, low net charge and a high tendency to form ß-structures. As the presence of such APRs is a prerequisite for aggregation, a plethora of methods have been developed to identify APRs in amino acid sequences. In the present chapter, the methodological basis of these approaches is discussed, as well as some practical applications.
Assuntos
Amiloide/química , Simulação por Computador , Modelos Moleculares , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Dobramento de Proteína , Análise de Sequência de ProteínaRESUMO
We investigated the origin and the effect of insertion D67D-THGERDLGPA within HIV-1 RT from a patient failing antiviral therapy. The insertion developed within the context of pre-existing NRTI and NNRTI mutations (M41L, L210W, T215Y and N348I). Concurrently, the NRTI mutations T69I and V118I and the NNRTI mutations K103N and Y181C were detected for the first time. High-level drug resistance (fold-changes≥50) and a good replication capacity (87% of wild-type) were observed, significantly higher than for the previous virus without insertion. The insertion was very similar to a region within human chromosome 17 (31/34 nucleotide identity), and had already been detected independently in a Japanese HIV-1 isolate. These results suggest that a particular sequence within human chromosome 17 is prone to horizontal gene transfer into the HIV-1 RT finger subdomain. This insertion confers selective advantage to HIV-1 by its contribution to multi-drug resistance and restoration of impaired replication capacity.
Assuntos
Farmacorresistência Viral , Transferência Genética Horizontal , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/genética , Replicação Viral , Cromossomos Humanos , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Conformação Proteica , RNA Viral/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Protein aggregation is geared by aggregation-prone regions that self-associate by ß-strand interactions. Charged residues and prolines are enriched at the flanks of aggregation-prone regions resulting in decreased aggregation. It is still unclear what drives the overrepresentation of these "aggregation gatekeepers", that is, whether their presence results from structural constraints determining protein stability or whether they constitute a bona fide functional class selectively maintained to control protein aggregation. As functional residues are typically conserved regardless of their cost to protein stability, we compared sequence conservation and thermodynamic cost of these residues in 2659 protein families in Escherichia coli. Across protein families, we find gatekeepers to be under strong selective conservation while at the same time representing a significant thermodynamic cost to protein structure. This finding supports the notion that aggregation gatekeepers are not structurally determined but evolutionary selected to control protein aggregation.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Desnaturação Proteica , Multimerização Proteica , Estabilidade Proteica , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
Aggregated forms of the amyloid-ß peptide are hypothesized to act as the prime toxic agents in Alzheimer disease (AD). The in vivo amyloid-ß peptide pool consists of both C- and N-terminally truncated or mutated peptides, and the composition thereof significantly determines AD risk. Other variations, such as biotinylation, are introduced as molecular tools to aid the understanding of disease mechanisms. Since these modifications have the potential to alter key aggregation properties of the amyloid-ß peptide, we present a comparative study of the aggregation of a substantial set of the most common in vivo identified and in vitro produced amyloid-ß peptides.