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1.
Cell ; 166(1): 193-208, 2016 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-27293189

RESUMEN

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.


Asunto(s)
Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/análisis , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Presenilina-2/análisis , Complejo 1 de Proteína Adaptadora/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Línea Celular Tumoral , Endosomas/química , Humanos , Lisosomas/química , Ratones , Presenilina-1/análisis , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratas , Especificidad por Sustrato
2.
Nucleic Acids Res ; 46(D1): D387-D392, 2018 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-29040693

RESUMEN

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.


Asunto(s)
Proteínas Amiloidogénicas/química , Bases de Datos de Proteínas , Interfaz Usuario-Computador
3.
Hum Mutat ; 40(9): 1530-1545, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31301157

RESUMEN

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.


Asunto(s)
Sustitución de Aminoácidos , Biología Computacional/métodos , Cistationina betasintasa/genética , Cistationina/metabolismo , Cistationina betasintasa/metabolismo , Homocisteína/metabolismo , Humanos , Fenotipo , Medicina de Precisión
4.
Mol Syst Biol ; 14(5): e8190, 2018 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-29759983

RESUMEN

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.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Activación Transcripcional , Clonación Molecular , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Aprendizaje Automático , Proteoma/genética , Análisis de Secuencia de ADN
5.
Acta Neuropathol ; 137(6): 901-918, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30874922

RESUMEN

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.


Asunto(s)
Inversión Cromosómica , Demencia/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/deficiencia , Mutación , Proteínas del Tejido Nervioso/deficiencia , Enfermedades Neurodegenerativas/genética , Neuronas/fisiología , Canales de Potasio/deficiencia , Potenciales de Acción/fisiología , Adulto , Anciano , Cromosomas Humanos Par 7/genética , Demencia/fisiopatología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Femenino , Genes Dominantes , Homeostasis , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Linaje , Penetrancia , Polimorfismo de Nucleótido Simple , Canales de Potasio/genética , Canales de Potasio/fisiología , Estabilidad Proteica , Transporte de Proteínas , Transmisión Sináptica , Secuenciación Completa del Genoma
6.
J Pathol ; 242(1): 24-38, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28035683

RESUMEN

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.


Asunto(s)
Neoplasias del Colon/genética , Glioblastoma/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Agregación Patológica de Proteínas/genética , Deficiencias en la Proteostasis/genética , Proteína p53 Supresora de Tumor/genética , Biopsia , Línea Celular Tumoral , Neoplasias del Colon/complicaciones , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citoplasma/metabolismo , Glioblastoma/complicaciones , Glioblastoma/metabolismo , Glioblastoma/patología , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Humanos , Estimación de Kaplan-Meier , Mutación , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/metabolismo , Deficiencias en la Proteostasis/etiología , Deficiencias en la Proteostasis/metabolismo , Receptores sigma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
7.
J Biol Chem ; 290(1): 242-58, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25391649

RESUMEN

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.


Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Lisosomas/metabolismo , Péptidos/metabolismo , Agregado de Proteínas , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Secuencia de Aminoácidos , Citocalasina D/farmacología , Proteínas de Unión al ADN/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Hidrazonas/farmacología , Concentración de Iones de Hidrógeno , Cinética , Lovastatina/farmacología , Lisosomas/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Pliegue de Proteína , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteolisis , Relación Estructura-Actividad , Factores de Transcripción/metabolismo
8.
Bioinformatics ; 31(15): 2580-2, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-25792555

RESUMEN

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.


Asunto(s)
Mutación/genética , Pliegue de Proteína , Proteínas/química , Proteínas/genética , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Bases de Datos de Proteínas , Humanos , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Proteínas/metabolismo , Termodinámica
9.
PLoS Comput Biol ; 11(9): e1004374, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26340370

RESUMEN

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.


Asunto(s)
Predisposición Genética a la Enfermedad/genética , Mutación/genética , Polimorfismo Genético/genética , Agregación Patológica de Proteínas/genética , Biología Computacional , Bases de Datos Factuales , Humanos , Estabilidad Proteica
10.
Nucleic Acids Res ; 40(Database issue): D935-9, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22075996

RESUMEN

Single nucleotide variants (SNVs) are, together with copy number variation, the primary source of variation in the human genome and are associated with phenotypic variation such as altered response to drug treatment and susceptibility to disease. Linking structural effects of non-synonymous SNVs to functional outcomes is a major issue in structural bioinformatics. The SNPeffect database (http://snpeffect.switchlab.org) uses sequence- and structure-based bioinformatics tools to predict the effect of protein-coding SNVs on the structural phenotype of proteins. It integrates aggregation prediction (TANGO), amyloid prediction (WALTZ), chaperone-binding prediction (LIMBO) and protein stability analysis (FoldX) for structural phenotyping. Additionally, SNPeffect holds information on affected catalytic sites and a number of post-translational modifications. The database contains all known human protein variants from UniProt, but users can now also submit custom protein variants for a SNPeffect analysis, including automated structure modeling. The new meta-analysis application allows plotting correlations between phenotypic features for a user-selected set of variants.


Asunto(s)
Bases de Datos de Proteínas , Polimorfismo de Nucleótido Simple , Conformación Proteica , Proteínas/genética , Humanos , Internet , Metaanálisis como Asunto , Fenotipo
11.
J Biol Chem ; 287(34): 28386-97, 2012 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-22773828

RESUMEN

Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression.


Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Chaperonas Moleculares/biosíntesis , Mutación Missense , alfa-Galactosidasa/metabolismo , 1-Desoxinojirimicina/farmacología , Activación Enzimática/efectos de los fármacos , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Chaperonas Moleculares/genética , alfa-Galactosidasa/genética
12.
J Biol Chem ; 287(44): 36732-43, 2012 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-22992745

RESUMEN

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aß(1-38) and Aß(1-40) at the cost of longer Aß peptides. Here, we consider the aggregation of Aß(1-38) and Aß(1-43) in addition to Aß(1-40) and Aß(1-42), in particular their behavior in mixtures representing the complex in vivo Aß pool. We demonstrate that Aß(1-38) and Aß(1-43) aggregate similar to Aß(1-40) and Aß(1-42), respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aß, Aß(1-38) and Aß(1-43) significantly affect the behaviors of Aß(1-40) and Aß(1-42). The short timescale conformational flexibility of Aß(1-38) is suggested to be responsible for enhancing toxicity of Aß(1-40) while exerting a cyto-protective effect on Aß(1-42). Our results indicate that the complex in vivo Aß peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.


Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/fisiología , Fragmentos de Péptidos/química , Multimerización de Proteína , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Amiloide/farmacología , Amiloide/ultraestructura , Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/fisiología , Benzotiazoles , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes/química , Humanos , Cinética , Microscopía de Fuerza Atómica , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/fisiología , Estructura Cuaternaria de Proteína , Tiazoles/química
13.
Cells ; 12(6)2023 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-36980299

RESUMEN

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.


Asunto(s)
Pliegue de Proteína , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Inhibidores de Proteasoma/farmacología , Respuesta al Choque Térmico , Bortezomib/farmacología
14.
PLoS Comput Biol ; 7(6): e1002090, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21731483

RESUMEN

We previously showed the existence of selective pressure against protein aggregation by the enrichment of aggregation-opposing 'gatekeeper' residues at strategic places along the sequence of proteins. Here we analyzed the relationship between protein lifetime and protein aggregation by combining experimentally determined turnover rates, expression data, structural data and chaperone interaction data on a set of more than 500 proteins. We find that selective pressure on protein sequences against aggregation is not homogeneous but that short-living proteins on average have a higher aggregation propensity and fewer chaperone interactions than long-living proteins. We also find that short-living proteins are more often associated to deposition diseases. These findings suggest that the efficient degradation of high-turnover proteins is sufficient to preclude aggregation, but also that factors that inhibit proteasomal activity, such as physiological ageing, will primarily affect the aggregation of short-living proteins.


Asunto(s)
Biología Computacional/métodos , Proteínas/química , Proteínas/metabolismo , Bases de Datos de Proteínas , Susceptibilidad a Enfermedades , Evolución Molecular , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana , Análisis por Matrices de Proteínas , Estabilidad Proteica , Proteínas/genética , Estadísticas no Paramétricas , Termodinámica , Factores de Tiempo
15.
Protein Eng Des Sel ; 32(10): 443-457, 2019 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-32399571

RESUMEN

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.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Mutación , Ingeniería de Proteínas , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Superóxido Dismutasa-1/química
16.
Protein Eng Des Sel ; 29(8): 285-9, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27284085

RESUMEN

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/.


Asunto(s)
Biología Computacional/métodos , Internet , Mutación , Agregado de Proteínas/genética , Proteínas/química , Proteínas/genética , Programas Informáticos , Algoritmos , Bases de Datos de Proteínas , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Termodinámica , Interfaz Usuario-Computador
17.
Nat Commun ; 7: 10816, 2016 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-26905391

RESUMEN

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.


Asunto(s)
Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Western Blotting , Línea Celular Tumoral , Cromatografía en Gel , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Mutación , Estabilidad Proteica , Solubilidad , alfa-Galactosidasa/química , alfa-Galactosidasa/genética
18.
Sci Rep ; 6: 20877, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26869068

RESUMEN

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.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Núcleo Celular/metabolismo , Dipéptidos/química , Drosophila melanogaster/genética , Demencia Frontotemporal/genética , Genes de Insecto , Pruebas Genéticas , Secuencias Repetitivas de Aminoácido , Transporte Activo de Núcleo Celular/genética , Animales , Arginina/metabolismo , Modelos Animales de Enfermedad , Ojo/patología , Células HeLa , Humanos , Metilación , Interferencia de ARN
19.
J Mol Biol ; 427(2): 236-47, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-25451783

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/genética , Agregado de Proteínas , Mapas de Interacción de Proteínas , beta-Galactosidasa/genética , Proteínas Bacterianas/metabolismo , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/genética , Unión Proteica/genética , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Espectroscopía Infrarroja por Transformada de Fourier , beta-Galactosidasa/metabolismo
20.
Neurology ; 85(24): 2116-25, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26581300

RESUMEN

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.


Asunto(s)
Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/genética , Mutación/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Bélgica/epidemiología , Estudios de Cohortes , Femenino , Demencia Frontotemporal/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Linaje
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