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1.
Parasitol Res ; 118(7): 2213-2221, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31183599

RESUMO

The Centrorhynchidae (Acanthocephala: Palaeacanthocephala) is a cosmopolitan family commonly found in various avian and mammalian hosts. Within Centrorhynchidae, species of the genus Sphaerirostris Golvan, 1956 are usually parasitic in the digestive tract of various passerine birds. In the present study, adult specimens of Sphaerirostris picae (Rudolphi, 1819), the type species of this genus, were recovered from the small intestine of Acridotheres tristis (Sturnidae) and Dendrocitta vagabunda (Corvidae) in Pakistan. Molecular data from the nuclear or mitochondrial genome is either very limited or completely absent from this phylogenetically understudied group of acanthocephalans. To fill this knowledge gap, we sequenced and determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) and the complete mitochondrial (mt) genome of S. picae. The ITS rDNA of S. picae was 95.2% similar to that of Sphaerirostris lanceoides which is the only member of the Centrorhynchidae whose ITS rDNA is available in GenBank. The phylogenetic tree based on the amino acid sequences of 12 mt protein-coding genes (PCGs) placed S. picae close to Centrorhynchus aluconis in a monophyletic clade of Polymorphida which also contain members of the families Polymorphidae and Plagiorhynchidae on separate branches. The mt gene arrangement, nucleotide composition and codon usage of 12 PCGs were discussed and compared with those of other acanthocephalan mt genomes. Within the Centrorhynchidae, S. picae and C. aluconis showed 67.7-86.8% similarity in the nucleotide sequences of 12 PCGs and 2 rRNAs, where nad4L is the most conserved gene while atp6 is the least conserved. The similarity in amino acid sequences ranged from 68.1 to 91.8%, where cox1 was recorded as the most conserved gene, while atp6 is highly variable among 12 PCGs. This novel mt genome of S. picae provides genetic resources for further studies of phylogenetics and molecular epidemiology of acanthocephalans.


Assuntos
Acantocéfalos/classificação , Acantocéfalos/genética , DNA Mitocondrial/genética , Genoma Mitocondrial/genética , Intestino Delgado/parasitologia , Passeriformes/parasitologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases , ATPases Transportadoras de Cálcio/genética , Ciclo-Oxigenase 1/genética , DNA Intergênico/genética , DNA de Protozoário/genética , DNA Ribossômico/genética , Paquistão , Filogenia , RNA Ribossômico/genética
2.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
3.
Biomed Res Int ; 2019: 2965035, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31073524

RESUMO

The type 2C protein which belongs to the major group of protein phosphatases (PP2C) plays a vital role in abscisic acid (ABA) signaling and signal transductions processes. In the present study, 131 PP2C genes were identified in total in Brassica rapa and categorized into thirteen subgroups based on their phylogenetic relationships. These B. rapa PP2C are structurally conserved based on amino acid sequence alignment, phylogenetic analysis, and conserved domains. Moreover, we utilized previously reported RNA-sequence data on various tissues (root, stem, leaf, flower, and silique), which suggests overlapping expression pattern in 29 paralogous gene pairs. The qRT-PCR validation of 15 paralogous gene pairs depicts distinct expression patterns in response to various abiotic stresses, such as heat, cold, ABA, and drought. Interestingly, stress-responsive BraPP2C candidate genes were also identified, suggesting their significance in stress-tolerance mechanism in B. rapa. The evolutionary analysis for 15 paralogous gene pairs suggested that only three pairs have the positive selection and remaining were purifying in nature. The presented results of this study hasten our understanding of the molecular evolution of the PP2C gene family in B. rapa. Thus, it will be ultimately helping in future research for facilitating the functional characterization of BraPP2C genes in developing the abiotic stress tolerant plants.


Assuntos
Brassica rapa/genética , Evolução Molecular , Filogenia , Proteína Fosfatase 2C/genética , Sequência de Aminoácidos/genética , Cromossomos de Plantas/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Família Multigênica/genética , Proteína Fosfatase 2C/classificação
4.
Mol Cell ; 74(5): 966-981.e18, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31078383

RESUMO

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.


Assuntos
Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos/genética , Humanos , Cinética , Ligação Proteica/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Ribossomos/química , Ribossomos/genética
5.
Arch Virol ; 164(8): 2201-2204, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31123964

RESUMO

In July 2018, a yellow mottle leaf disease was observed on the leaves of Euonymus bungeanus Maxim plants in Liaoning Province of China. Typical flexuous filaments (diameter, 13 nm; length, ca. 500 nm) were observed in extracts on the symptomatic leaves. Transcriptome sequencing indicated the presence of a potexvirus in the infected samples. The complete viral genome, determined to be 6,784 nucleotides in length, excluding the poly(A) tail, contains five open reading frames and is most closely related to that of euonymus yellow vein associated virus (EuYVAV, MF078061) (41.0%). Based on the coat protein gene, this virus shares the highest sequence similarity with cymbidium mosaic virus (CyMV, EF125179), with 43.9% nucleotide and 38.8% amino acid sequence identity, respectively. Phylogenetic analysis indicated that the virus clustered with potexviruses and is most closely related to EuYVaV. This virus is a distinct member of the genus Potexvirus, for which the name "euonymus yellow mottle associated virus" (EuYMaV) is proposed.


Assuntos
Euonymus/virologia , Genoma Viral/genética , Potexvirus/genética , RNA Viral/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas do Capsídeo/genética , China , Fases de Leitura Aberta/genética , Filogenia , Doenças das Plantas/virologia , Folhas de Planta/virologia , Plantas/virologia , Vírus Satélites/genética , Análise de Sequência de RNA/métodos
6.
Parasitol Res ; 118(6): 1811-1820, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31049696

RESUMO

The larval stages of the tapeworm Echinococcus granulosus (Cestoda: Taeniidae) are the causative agent of cystic echinococcosis, one of the most important parasitic zoonoses worldwide. E. granulosus has a complete pathway for the tricarboxylic acid cycle (TCA), in which citrate synthase (CS) is the key enzyme. Here, we cloned and expressed CS from E. granulosus (Eg-CS) and report its molecular characterization. The localization of this protein during different developmental stages and mRNA expression patterns during H2O2 treatment were determined. We found that Eg-CS is a highly conserved protein, consisting of 466 amino acids. In western blotting assays, recombinant Eg-CS (rEg-CS) reacted with E. granulosus-positive sheep sera and anti-rEg-CS rabbit sera, indicating that Eg-CS has good antigenicity and immunoreactivity. Localization studies, performed using immunohistochemistry, showed that Eg-CS is ubiquitously expressed in the larva, germinal layer, and adult worm sections of E. granulosus. Eg-CS mRNA expression levels increased following H2O2 exposure. In conclusion, citrate synthase might be involved in the metabolic process in E. granulosus. An assessment of the serodiagnostic potential of rEg-CS based on indirect ELISA showed that, although sensitivity (93.55%) and specificity (80.49%) are high, cross-reactivity with other parasites precludes its use as a diagnostic antigen.


Assuntos
Citrato (si)-Sintase/genética , Equinococose/diagnóstico , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Larva/metabolismo , Sequência de Aminoácidos/genética , Animais , Anticorpos/imunologia , Western Blotting , Citrato (si)-Sintase/imunologia , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico/genética , Clonagem Molecular , Reações Cruzadas/imunologia , Equinococose/parasitologia , Ensaio de Imunoadsorção Enzimática , Peróxido de Hidrogênio/metabolismo , Coelhos , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Ovinos/genética , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/parasitologia
7.
Gene ; 708: 57-62, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31128224

RESUMO

Zika virus (ZIKV) has spread globally and has been linked to the onset of microcephaly and other brain abnormalities. The ZIKV genome consists of an ~10.7 kb positive-stranded RNA molecule that encodes three structural (C, prM and E) and seven nonstructural (5'-NS1-NS2A-NS2B-NS3- NS4A/2K-NS4B-NS5-3') proteins. In this work, we looked for genetic variants in 485 ZIKV complete genomes from GenBank (NCBI) and performed a computational systematic approach using MAESTROweb server to assess the impact of nonsynonymous mutations in ZIKV proteins (C, M, E, NS1, NS2A, NS2B-NS3 protease, NS3 helicase and NS5). Then, we merged the data and correlated it with the phenotypic reports of ZIKV circulating strains. The sensitivity profile of the proteins showed 96 mutational hotspots. We found 22 relevant mutations in proteins C (I80T), NS2A (I34M/T/V, I45V, I80T/V, L113F, A117V, I118V, L128P, V143A, T151A, M199I/V, R207K and L208I) and NS3 helicase (D436G, Y498H, R525K, Q528R and R583K) of the circulating strains. Our analysis exploited the impact of nonsynonymous mutations on ZIKV proteins, their structural and functional insights. The results presented here could advance our current understanding on ZIKV proteins functions and pathogenesis.


Assuntos
Genoma Viral/genética , Proteínas Virais/genética , Zika virus/genética , Sequência de Aminoácidos/genética , Mutação , Conformação Proteica , Zika virus/patogenicidade
8.
Nat Commun ; 10(1): 2013, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043592

RESUMO

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.


Assuntos
Sequência de Aminoácidos/genética , Regulação da Expressão Gênica , Engenharia de Proteínas/métodos , Proteólise , Biotecnologia/métodos , Células HEK293 , Meia-Vida , Células HeLa , Humanos , Microscopia Intravital/métodos , Células-Tronco Mesenquimais , Microscopia de Fluorescência , Biologia Sintética/métodos
9.
Mol Cell ; 74(5): 909-921.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006538

RESUMO

Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Retículo Endoplasmático/genética , Proteínas Intrinsicamente Desordenadas/genética , Sequência de Aminoácidos/genética , Proteínas Relacionadas à Autofagia/química , Proteínas de Ciclo Celular/genética , Retículo Endoplasmático/química , Estresse do Retículo Endoplasmático/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteólise
10.
Nucleic Acids Res ; 47(7): 3699-3710, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30993346

RESUMO

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.


Assuntos
RNA Helicases DEAD-box/química , RNA/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos/genética , RNA Helicases DEAD-box/genética , Humanos , Fenômenos Mecânicos , RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-30961818

RESUMO

This study aimed to investigate the prevalence of influenza A viruses in birds and humans residing in the same localities of Sharkia Province, Egypt and the risk factors' assessment in poultry farms. A total of 100 birds comprised of 50 chickens, 25 ducks and 25 wild egrets were sampled. Swab samples were collected from 65 people (50 poultry farm workers and 15 hospitalized patients). All samples were screened for the presence of influenza A viruses using isolation and molecular assays. Avian influenza viruses were only detected in chicken samples (18%) and molecularly confirmed as subtype H5. The infection rate was higher in broilers (40%) than layers (8.6%). Influenza A (H1) pdm09 virus was detected in a single human case (1.54%). All the isolated AI H5 viruses were clustered into clade (2.2.1.2) and shared a high similarity rate at nucleotides and amino acid levels. In addition, they had a multi-basic amino acid motif (ـــPQGEKRRKKR/GLFـــ) at the H5 gene cleavage site that exhibited point mutations. Chicken breed, movement of workers from one flock to another, lack of utensils' disinfection and the introduction of new birds to the farm were significant risk factors associated with highly pathogenic AI H5 virus infection in poultry farms (p ≤ 0.05). Other factors showed no significant association. The HPAI H5 viruses are still endemic in Egypt with continuous mutation. Co-circulation of these viruses in birds and pdm09 viruses in humans raises alarm for the emergence of reassortant viruses that are capable of potentiating pandemics.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Sequência de Aminoácidos/genética , Animais , Galinhas/virologia , Patos/virologia , Egito/epidemiologia , Fazendas , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Masculino , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Prevalência , Fatores de Risco
12.
Methods Mol Biol ; 1964: 3-15, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929231

RESUMO

Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating (CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all share the common property of interacting with the cellular lipid membrane in their mode of action. Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help decipher their mechanism of action. Among the different biophysical methods that can be used to fully characterize P/L interactions, differential scanning calorimetry (DSC) allows determining the peptide effect on the lipid phase transitions, a property that reflects the P/L interaction mode. A general protocol for classical DSC experiments for P/L studies will be provided.


Assuntos
Varredura Diferencial de Calorimetria/métodos , Peptídeos Penetradores de Células/química , Lipídeos de Membrana/química , Biologia Molecular/métodos , Sequência de Aminoácidos/genética , Peptídeos Penetradores de Células/genética , Dicroísmo Circular , Bicamadas Lipídicas/química , Lipídeos de Membrana/genética , Transição de Fase
13.
Methods Mol Biol ; 1958: 15-45, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945212

RESUMO

Due to the advancement in various sequencing technologies, the gap between the number of protein sequences and the number of experimental protein structures is ever increasing. Community-wide initiatives like CASP have resulted in considerable efforts in the development of computational methods to accurately model protein structures from sequences. Sequence-based prediction of super-secondary structure has direct application in protein structure prediction, and there have been significant efforts in the prediction of super-secondary structure in the last decade. In this chapter, we first introduce the protein structure prediction problem and highlight some of the important progress in the field of protein structure prediction. Next, we discuss recent methods for the prediction of super-secondary structures. Finally, we discuss applications of super-secondary structure prediction in structure prediction/analysis of proteins. We also discuss prediction of protein structures that are composed of simple super-secondary structure repeats and protein structures that are composed of complex super-secondary structure repeats. Finally, we also discuss the recent trends in the field.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Dobramento de Proteína , Proteínas/química , Sequência de Aminoácidos/genética , Biologia Computacional/tendências , Humanos , Conformação Proteica
14.
Methods Mol Biol ; 1958: 73-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945214

RESUMO

Many new methods for the sequence-based prediction of the secondary and supersecondary structures have been developed over the last several years. These and older sequence-based predictors are widely applied for the characterization and prediction of protein structure and function. These efforts have produced countless accurate predictors, many of which rely on state-of-the-art machine learning models and evolutionary information generated from multiple sequence alignments. We describe and motivate both types of predictions. We introduce concepts related to the annotation and computational prediction of the three-state and eight-state secondary structure as well as several types of supersecondary structures, such as ß hairpins, coiled coils, and α-turn-α motifs. We review 34 predictors focusing on recent tools and provide detailed information for a selected set of 14 secondary structure and 3 supersecondary structure predictors. We conclude with several practical notes for the end users of these predictive methods.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Proteínas/química , Alinhamento de Sequência/métodos , Algoritmos , Sequência de Aminoácidos/genética , Dobramento de Proteína
15.
Methods Mol Biol ; 1958: 101-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945215

RESUMO

Supersecondary structure (SSS) refers to specific geometric arrangements of several secondary structure (SS) elements that are connected by loops. The SSS can provide useful information about the spatial structure and function of a protein. As such, the SSS is a bridge between the secondary structure and tertiary structure. In this chapter, we propose a stacking-based machine learning method for the prediction of two types of SSSs, namely, ß-hairpins and ß-α-ß, from the protein sequence based on comprehensive feature encoding. To encode protein residues, we utilize key features such as solvent accessibility, conservation profile, half surface exposure, torsion angle fluctuation, disorder probabilities, and more. The usefulness of the proposed approach is assessed using a widely used threefold cross-validation technique. The obtained empirical result shows that the proposed approach is useful and prediction can be improved further.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Proteínas/química , Algoritmos , Sequência de Aminoácidos/genética , Bases de Dados de Proteínas , Modelos Moleculares , Proteínas/genética , Software
16.
Methods Mol Biol ; 1958: 221-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945221

RESUMO

ßαß super-secondary structures constitute the basic building blocks of (ß/α)8 class of proteins. Despite the success in designing super-secondary structures, till date, there is not a single example of a natural ßαß sequence known to fold in isolation. In this chapter, to address the finding the "needles" in the haystack scenario, we have combined the sequence preferences and structural features of independent ßαß motifs, dictated by natural selection, with rationally derived parameters from a designed ßαß motif adopting stable fold in solution. Guided by this approach, a set of potential ßαß sequences from (ß/α)8/TIM barrels are proposed as likely candidates for autonomously folding based on the assessment of their foldability.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Dobramento de Proteína , Proteínas/química , Sequência de Aminoácidos/genética , Ligações de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Proteínas/genética
17.
Methods Mol Biol ; 1958: 237-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945222

RESUMO

Proteins with prion-like behavior are attracting an increasing interest, since accumulating evidences indicate that they play relevant roles both in health and disease. The self-assembly of these proteins into insoluble aggregates is associated with severe neuropathological processes such as amyotrophic lateral sclerosis (ALS). However, in normal conditions, they are known to accomplish a wide range of functional roles. The conformational duality of prion-like proteins is often encoded in specific protein regions, named prion-like domains (PrLDs). PrLDs are usually long and disordered regions of low complexity. We have shown that PrLDs might contain soft-amyloid cores that contribute significantly to trigger their aggregation, as well as to support their propagation. Further exploration of the role of these sequences in the conformational conversion of prion-like proteins might provide novel insights into the mechanism of action and regulation of these polypeptides, enabling the future development of therapeutic strategies. Here, we describe a set of methodologies aimed to identify and characterize these short amyloid stretches in a protein or proteome of interest, ranging from in silico detection to in vitro and in vivo evaluation and validation.


Assuntos
Biologia Molecular/métodos , Proteínas Priônicas/química , Príons/química , Sequência de Aminoácidos/genética , Amiloide/química , Amiloide/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Proteínas Priônicas/genética , Príons/genética , Agregados Proteicos/genética , Domínios Proteicos/genética , Proteoma/química , Proteoma/genética
18.
Methods Mol Biol ; 1958: 297-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945225

RESUMO

A relatively recently discovered class of proteins known as transformer proteins undergo large-scale conformational conversions that change their supersecondary structure. These structural transformations lead to different configurations that perform different functions. We describe computational methods using molecular dynamics simulations that allow the determination of the specific amino acids that facilitate the conformational transformations. These investigations provide guidance on the location and type of amino acid mutations that can either enhance or inhibit the structural transitions that allow transformer proteins to perform multiple functions.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Proteínas/química , Sequência de Aminoácidos/genética , Simulação de Dinâmica Molecular , Mutação/genética , Multimerização Proteica , Proteínas/genética
19.
Methods Mol Biol ; 1958: 313-327, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945226

RESUMO

The goal is to define sequence characteristics of beta-sandwich proteins that are unique for the beta-sandwich supersecondary structure (SSS). Finding of the conserved residues that are critical for protein structure can often be accomplished with homology methods, but these methods are not always adequate as residues with similar structural role do not always occupy the same position as determined by sequence alignment. In this paper, we show how to identify residues that play the same structural role in the different proteins of the same SSS, even when these residue positions cannot be aligned with sequence alignment methods. The SSS characteristics are (a) a set of positions in each strand that are involved in the formation of a hydrophobic core, residue content, and correlations of residues at these key positions, (b) maximum allowable number of "low-frequency residues" for each strand, (c) minimum allowed number of "high-frequency" residues for each loop, and (d) minimum and maximum lengths of each loop. These sequence characteristics are referred to as "sequence pattern" for their respective SSS. The high specificity and sensitivity for a particular SSS are confirmed by applying this pattern to all protein structures in the SCOP data bank. We present here the pattern for one of the most common SSS of beta-sandwich proteins.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Proteínas/química , Alinhamento de Sequência/métodos , Algoritmos , Sequência de Aminoácidos/genética , Bases de Dados de Proteínas , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Software
20.
Methods Mol Biol ; 1958: 329-340, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945227

RESUMO

Supersecondary structure code (SSSC), which is represented as the combination of α-helix-type (SSSC: H), ß-sheet-type (SSSC: S), the other (SSSC: T), and disorder residue or C-terminal (SSSC: D) patterns, has been produced by the developed concept of Ramachandran plot, in addition, with the ω angle and with the specification of positions of torsion angles in a protein by the registration of codes for torsion angles of each amino acid peptide unit, derived from the fuzzy search of structural code homology using the template patterns 3a5c4a (SSSC: H) and 6c4a4a (SSSC: S) with conformational codes. The DSSP (Dictionary of Secondary Structure in Proteins) method assigns the secondary structure including hydrogen bond well. In contrast, supersecondary structure code is very sensitive to the supersecondary structures of proteins. In this chapter, the protocol of homology search methods, the sequence alignment using supersecondary structure code, the assignment of supersecondary structure code T, the fuzzy search using supersecondary structure code, and the exact search using supersecondary structure code are described. Supersecondary structure code is variable with the conformational change. If possible, many Protein Data Bank (PDB) data of similar main chains of proteins should be used for the homology searches. The thorough check of SSSC sequences is also useful to reveal the role of target pattern.


Assuntos
Motivos de Aminoácidos , Proteínas/química , Homologia de Sequência de Aminoácidos , Software , Sequência de Aminoácidos/genética , Bases de Dados de Proteínas , Ligações de Hidrogênio , Modelos Moleculares , Peptídeos/química , Proteínas/genética , Alinhamento de Sequência/métodos
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