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1.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-33800121

RESUMEN

Nitrotyrosine, which is generated by numerous reactive nitrogen species, is a type of protein post-translational modification. Identification of site-specific nitration modification on tyrosine is a prerequisite to understanding the molecular function of nitrated proteins. Thanks to the progress of machine learning, computational prediction can play a vital role before the biological experimentation. Herein, we developed a computational predictor PredNTS by integrating multiple sequence features including K-mer, composition of k-spaced amino acid pairs (CKSAAP), AAindex, and binary encoding schemes. The important features were selected by the recursive feature elimination approach using a random forest classifier. Finally, we linearly combined the successive random forest (RF) probability scores generated by the different, single encoding-employing RF models. The resultant PredNTS predictor achieved an area under a curve (AUC) of 0.910 using five-fold cross validation. It outperformed the existing predictors on a comprehensive and independent dataset. Furthermore, we investigated several machine learning algorithms to demonstrate the superiority of the employed RF algorithm. The PredNTS is a useful computational resource for the prediction of nitrotyrosine sites. The web-application with the curated datasets of the PredNTS is publicly available.


Asunto(s)
Biología Computacional , Aprendizaje Automático , Procesamiento Proteico-Postraduccional , Proteínas/genética , Análisis de Secuencia de Proteína , Máquina de Vectores de Soporte , Tirosina/análogos & derivados , Tirosina/genética
2.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-33803345

RESUMEN

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Regulación de la Expresión Génica , Alcaloides/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Cicloheximida/farmacología , Células HEK293 , Humanos , Leupeptinas/farmacología , Proteínas/genética , Proteínas/metabolismo
3.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33809039

RESUMEN

Taking advantage of the last cryogenic electron microscopy structure of human huntingtin, we explored with computational methods its physicochemical properties, focusing on the solvent accessible surface of the protein and highlighting a quite interesting mix of hydrophobic and hydrophilic patterns, with the prevalence of the latter ones. We then evaluated the probability of exposed residues to be in contact with other proteins, discovering that they tend to cluster in specific regions of the protein. We then found that the remaining portions of the protein surface can contain calcium-binding sites that we propose here as putative mediators for the protein to interact with membranes. Our findings are justified in relation to the present knowledge of huntingtin functional annotation.


Asunto(s)
Calcio/metabolismo , Biología Computacional , Proteína Huntingtina/química , Proteínas/genética , Sitios de Unión/genética , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica/genética , Solventes/química , Propiedades de Superficie
4.
Nat Commun ; 12(1): 2046, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824347

RESUMEN

Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Huesos/patología , Proteínas/metabolismo , Esclerosis/patología , Transducción de Señal , Proteínas Smad/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Núcleo Celular/metabolismo , Niño , Preescolar , Femenino , Humanos , Masculino , Ratones Mutantes , Mutación/genética , Osteoblastos/patología , Linaje , Fosforilación , Proteínas/genética , Cráneo/patología , Adulto Joven
5.
Nat Commun ; 12(1): 2200, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33850130

RESUMEN

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.


Asunto(s)
Inteínas/fisiología , Ingeniería de Proteínas/métodos , Proteínas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Inteínas/genética , Modelos Moleculares , Conformación Proteica , Empalme de Proteína , Proteínas/química , Proteínas/genética , Biología Sintética/métodos
6.
BMC Bioinformatics ; 22(1): 171, 2021 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-33789579

RESUMEN

BACKGROUND: Protein post-translational modification (PTM) is a key issue to investigate the mechanism of protein's function. With the rapid development of proteomics technology, a large amount of protein sequence data has been generated, which highlights the importance of the in-depth study and analysis of PTMs in proteins. METHOD: We proposed a new multi-classification machine learning pipeline MultiLyGAN to identity seven types of lysine modified sites. Using eight different sequential and five structural construction methods, 1497 valid features were remained after the filtering by Pearson correlation coefficient. To solve the data imbalance problem, Conditional Generative Adversarial Network (CGAN) and Conditional Wasserstein Generative Adversarial Network (CWGAN), two influential deep generative methods were leveraged and compared to generate new samples for the types with fewer samples. Finally, random forest algorithm was utilized to predict seven categories. RESULTS: In the tenfold cross-validation, accuracy (Acc) and Matthews correlation coefficient (MCC) were 0.8589 and 0.8376, respectively. In the independent test, Acc and MCC were 0.8549 and 0.8330, respectively. The results indicated that CWGAN better solved the existing data imbalance and stabilized the training error. Alternatively, an accumulated feature importance analysis reported that CKSAAP, PWM and structural features were the three most important feature-encoding schemes. MultiLyGAN can be found at https://github.com/Lab-Xu/MultiLyGAN . CONCLUSIONS: The CWGAN greatly improved the predictive performance in all experiments. Features derived from CKSAAP, PWM and structure schemes are the most informative and had the greatest contribution to the prediction of PTM.


Asunto(s)
Lisina , Procesamiento Proteico-Postraduccional , Proteínas , Algoritmos , Lisina/metabolismo , Aprendizaje Automático , Proteínas/genética
7.
Nat Methods ; 18(3): 233, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33674794
8.
BMC Bioinformatics ; 22(1): 121, 2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33711918

RESUMEN

BACKGROUND: The identification of protein families is of outstanding practical importance for in silico protein annotation and is at the basis of several bioinformatic resources. Pfam is possibly the most well known protein family database, built in many years of work by domain experts with extensive use of manual curation. This approach is generally very accurate, but it is quite time consuming and it may suffer from a bias generated from the hand-curation itself, which is often guided by the available experimental evidence. RESULTS: We introduce a procedure that aims to identify automatically putative protein families. The procedure is based on Density Peak Clustering and uses as input only local pairwise alignments between protein sequences. In the experiment we present here, we ran the algorithm on about 4000 full-length proteins with at least one domain classified by Pfam as belonging to the Pseudouridine synthase and Archaeosine transglycosylase (PUA) clan. We obtained 71 automatically-generated sequence clusters with at least 100 members. While our clusters were largely consistent with the Pfam classification, showing good overlap with either single or multi-domain Pfam family architectures, we also observed some inconsistencies. The latter were inspected using structural and sequence based evidence, which suggested that the automatic classification captured evolutionary signals reflecting non-trivial features of protein family architectures. Based on this analysis we identified a putative novel pre-PUA domain as well as alternative boundaries for a few PUA or PUA-associated families. As a first indication that our approach was unlikely to be clan-specific, we performed the same analysis on the P53 clan, obtaining comparable results. CONCLUSIONS: The clustering procedure described in this work takes advantage of the information contained in a large set of pairwise alignments and successfully identifies a set of putative families and family architectures in an unsupervised manner. Comparison with the Pfam classification highlights significant overlap and points to interesting differences, suggesting that our new algorithm could have potential in applications related to automatic protein classification. Testing this hypothesis, however, will require further experiments on large and diverse sequence datasets.


Asunto(s)
Proteínas , Alineación de Secuencia , Secuencia de Aminoácidos , Análisis por Conglomerados , Bases de Datos de Proteínas , Humanos , Proteínas/genética
9.
Lancet Diabetes Endocrinol ; 9(4): 235-246, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33647242

RESUMEN

Prader-Willi syndrome is a rare genetic neurodevelopmental disorder resulting from the loss of expression of maternally imprinted genes located in the paternal chromosomal region, 15q11-13. Impaired hypothalamic development and function is the cause of most of the phenotypes comprising the developmental trajectory of Prader-Willi syndrome: from anorexia at birth to excessive weight gain preceding hyperphagia, and early severe obesity with hormonal deficiencies, behavioural problems, and dysautonomia. Growth hormone deficiency, hypogonadism, hypothyroidism, premature adrenarche, corticotropin deficiency, precocious puberty, and glucose metabolism disorders are the main endocrine dysfunctions observed. Additionally, as a result of hypothalamic dysfunction, oxytocin and ghrelin systems are impaired in most patients. Standard pituitary and gonadal hormone replacement therapies are required. In this Review, we discuss Prader-Willi syndrome as a model of hypothalamic dysfunction, and provide a comprehensive description of the accumulated knowledge on genetics, pathophysiology, and treatment approaches of this rare disorder.


Asunto(s)
Enfermedades del Sistema Endocrino/fisiopatología , Hipotálamo/fisiopatología , Síndrome de Prader-Willi/fisiopatología , Animales , Enfermedades del Sistema Endocrino/genética , Enfermedades del Sistema Endocrino/terapia , Humanos , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/terapia , Proteínas/genética
10.
Molecules ; 26(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673040

RESUMEN

Background: Zinc binding proteins make up a significant proportion of the proteomes of most organisms and, within those proteins, zinc performs rôles in catalysis and structure stabilisation. Identifying the ability to bind zinc in a novel protein can offer insights into its functions and the mechanism by which it carries out those functions. Computational means of doing so are faster than spectroscopic means, allowing for searching at much greater speeds and scales, and thereby guiding complimentary experimental approaches. Typically, computational models of zinc binding predict zinc binding for individual residues rather than as a single binding site, and typically do not distinguish between different classes of binding site-missing crucial properties indicative of zinc binding. Methods: Previously, we created ZincBindDB, a continuously updated database of known zinc binding sites, categorised by family (the set of liganding residues). Here, we use this dataset to create ZincBindPredict, a set of machine learning methods to predict the most common zinc binding site families for both structure and sequence. Results: The models all achieve an MCC ≥ 0.88, recall ≥ 0.93 and precision ≥ 0.91 for the structural models (mean MCC = 0.97), while the sequence models have MCC ≥ 0.64, recall ≥ 0.80 and precision ≥ 0.83 (mean MCC = 0.87), with the models for binding sites containing four liganding residues performing much better than this. Conclusions: The predictors outperform competing zinc binding site predictors and are available online via a web interface and a GraphQL API.


Asunto(s)
Biología Computacional , Proteínas/química , Programas Informáticos , Zinc/química , Algoritmos , Sitios de Unión/genética , Bases de Datos de Proteínas , Ligandos , Aprendizaje Automático , Unión Proteica/genética , Proteínas/genética , Máquina de Vectores de Soporte
11.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669299

RESUMEN

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.


Asunto(s)
Eliminación de Gen , Ingeniería Genética/métodos , Hanseniaspora/enzimología , Hanseniaspora/genética , Microorganismos Modificados Genéticamente/genética , Proteínas/genética , Acetatos/metabolismo , Alelos , Fermentación/genética , Genes Fúngicos , Sistemas de Lectura Abierta , Fenotipo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vitis/metabolismo , Vino
12.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669425

RESUMEN

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.


Asunto(s)
Acrosoma/patología , Fertilización In Vitro/métodos , Eliminación de Gen , Infertilidad Masculina/genética , Proteínas/genética , Acrosoma/metabolismo , Acrosoma/ultraestructura , Animales , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Femenino , Infertilidad Masculina/metabolismo , Gotas Lipídicas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Embarazo , Proteínas/metabolismo , Motilidad Espermática/genética , Espermatogénesis/genética , Testículo/metabolismo
13.
BMC Bioinformatics ; 22(1): 162, 2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771095

RESUMEN

BACKGROUND: Hidden Markov models (HMM) are a powerful tool for analyzing biological sequences in a wide variety of applications, from profiling functional protein families to identifying functional domains. The standard method used for HMM training is either by maximum likelihood using counting when sequences are labelled or by expectation maximization, such as the Baum-Welch algorithm, when sequences are unlabelled. However, increasingly there are situations where sequences are just partially labelled. In this paper, we designed a new training method based on the Baum-Welch algorithm to train HMMs for situations in which only partial labeling is available for certain biological problems. RESULTS: Compared with a similar method previously reported that is designed for the purpose of active learning in text mining, our method achieves significant improvements in model training, as demonstrated by higher accuracy when the trained models are tested for decoding with both synthetic data and real data. CONCLUSIONS: A novel training method is developed to improve the training of hidden Markov models by utilizing partial labelled data. The method will impact on detecting de novo motifs and signals in biological sequence data. In particular, the method will be deployed in active learning mode to the ongoing research in detecting plasmodesmata targeting signals and assess the performance with validations from wet-lab experiments.


Asunto(s)
Algoritmos , Proteínas , Biología Computacional , Cadenas de Markov , Proteínas/genética
14.
Nat Genet ; 53(3): 313-321, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33664507

RESUMEN

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.


Asunto(s)
Variación Genética , Células Madre Pluripotentes Inducidas/fisiología , Sitios de Carácter Cuantitativo , Síndrome de Bardet-Biedl/genética , Canales de Calcio/genética , Línea Celular , Ataxia Cerebelosa/genética , Metilación de ADN , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Polimorfismo de Nucleótido Simple , Proteínas/genética , Enfermedades Raras/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN , Secuenciación Completa del Genoma
15.
Mol Syst Biol ; 17(3): e9923, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33749993

RESUMEN

Molecular knowledge of biological processes is a cornerstone in omics data analysis. Applied to single-cell data, such analyses provide mechanistic insights into individual cells and their interactions. However, knowledge of intercellular communication is scarce, scattered across resources, and not linked to intracellular processes. To address this gap, we combined over 100 resources covering interactions and roles of proteins in inter- and intracellular signaling, as well as transcriptional and post-transcriptional regulation. We added protein complex information and annotations on function, localization, and role in diseases for each protein. The resource is available for human, and via homology translation for mouse and rat. The data are accessible via OmniPath's web service (https://omnipathdb.org/), a Cytoscape plug-in, and packages in R/Bioconductor and Python, providing access options for computational and experimental scientists. We created workflows with tutorials to facilitate the analysis of cell-cell interactions and affected downstream intracellular signaling processes. OmniPath provides a single access point to knowledge spanning intra- and intercellular processes for data analysis, as we demonstrate in applications studying SARS-CoV-2 infection and ulcerative colitis.


Asunto(s)
/metabolismo , Colitis Ulcerosa/metabolismo , Biología Computacional/métodos , Proteínas/metabolismo , Transducción de Señal , Animales , Comunicación Celular , Colitis Ulcerosa/patología , Bases de Datos Factuales , Enzimas/metabolismo , Humanos , Ratones , Procesamiento Proteico-Postraduccional , Proteínas/genética , Ratas , Análisis de la Célula Individual , Programas Informáticos , Flujo de Trabajo
16.
Nat Commun ; 12(1): 1515, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750777

RESUMEN

Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5'UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.


Asunto(s)
Enfermedad/genética , Sistemas de Lectura Abierta , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Genoma Humano , Humanos , Fenotipo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas/genética , Receptor EphB2
17.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652889

RESUMEN

Genetic code expansion (GCE) technology is a useful tool for the site-specific modification of proteins. An unnatural amino acid (UAA) is one of the essential components of this technique, typically required at high concentration (1 mM or higher) in growth medium. The supply of UAAs is an important limitation to the application of GCE technology, as many UAAs are either expansive or commercially unavailable. In this study, two UAAs in a racemic mixture were converted into optically pure forms using two enzymes, the d-amino acid oxidase (RgDAAO) from Rhodotorula gracilis and the aminotransferase (TtAT) from Thermus thermophilus. In the coupled enzyme system, RgDAAO oxidizes the d-form of UAAs in a stereospecific manner and produces the corresponding α-keto acids, which are then converted into the l-form of UAAs by TtAT, resulting in the quantitative and stereospecific conversion of racemic UAAs to optically pure forms. The genetic incorporation of the optically pure UAAs into a target protein produced a better protein yield than the same experiments using the racemic mixtures of the UAAs. This method could not only be used for the preparation of optically pure UAAs from racemic mixtures, but also the broad substrate specificity of both enzymes would allow for its expansion to structurally diverse UAAs.


Asunto(s)
Aminoácidos/genética , Ingeniería de Proteínas , Proteínas/genética , Rhodotorula/genética , Aminoácidos/química , Clonación Molecular , Medios de Cultivo/química , Escherichia coli/genética , Código Genético , Proteínas/química , Rhodotorula/química , Especificidad por Sustrato
18.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652992

RESUMEN

Substances that can modify the androgen receptor pathway in humans and animals are entering the environment and food chain with the proven ability to disrupt hormonal systems and leading to toxicity and adverse effects on reproduction, brain development, and prostate cancer, among others. State-of-the-art databases with experimental data of human, chimp, and rat effects by chemicals have been used to build machine-learning classifiers and regressors and to evaluate these on independent sets. Different featurizations, algorithms, and protein structures lead to different results, with deep neural networks (DNNs) on user-defined physicochemically relevant features developed for this work outperforming graph convolutional, random forest, and large featurizations. The results show that these user-provided structure-, ligand-, and statistically based features and specific DNNs provided the best results as determined by AUC (0.87), MCC (0.47), and other metrics and by their interpretability and chemical meaning of the descriptors/features. In addition, the same features in the DNN method performed better than in a multivariate logistic model: validation MCC = 0.468 and training MCC = 0.868 for the present work compared to evaluation set MCC = 0.2036 and training set MCC = 0.5364 for the multivariate logistic regression on the full, unbalanced set. Techniques of this type may improve AR and toxicity description and prediction, improving assessment and design of compounds. Source code and data are available on github.


Asunto(s)
Aprendizaje Profundo , Unión Proteica/genética , Proteínas/genética , Receptores Androgénicos/genética , Algoritmos , Animales , Humanos , Ligandos , Modelos Logísticos , Redes Neurales de la Computación , Ratas , Programas Informáticos
19.
Nat Commun ; 12(1): 1745, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741971

RESUMEN

Hydrogen sulfide (H2S) is a cytoprotective redox-active metabolite that signals through protein persulfidation (R-SSnH). Despite the known importance of persulfidation, tissue-specific persulfidome profiles and their associated functions are not well characterized, specifically under conditions and interventions known to modulate H2S production. We hypothesize that dietary restriction (DR), which increases lifespan and can boost H2S production, expands tissue-specific persulfidomes. Here, we find protein persulfidation enriched in liver, kidney, muscle, and brain but decreased in heart of young and aged male mice under two forms of DR, with DR promoting persulfidation in numerous metabolic and aging-related pathways. Mice lacking cystathionine γ-lyase (CGL) have overall decreased tissue protein persulfidation numbers and fail to functionally augment persulfidomes in response to DR, predominantly in kidney, muscle, and brain. Here, we define tissue- and CGL-dependent persulfidomes and how diet transforms their makeup, underscoring the breadth for DR and H2S to impact biological processes and organismal health.


Asunto(s)
Cistationina gamma-Liasa/química , Cistationina gamma-Liasa/metabolismo , Dieta , Proteínas/química , Proteínas/metabolismo , Envejecimiento/metabolismo , Animales , Encéfalo/metabolismo , Cistationina gamma-Liasa/genética , Sulfuro de Hidrógeno/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Longevidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculos/metabolismo , Proteínas/genética , Transcriptoma
20.
Nat Commun ; 12(1): 1809, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753744

RESUMEN

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
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