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1.
BMC Bioinformatics ; 22(1): 320, 2021 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-34118870

RESUMEN

BACKGROUND: Assignment of chemical compounds to biological pathways is a crucial step to understand the relationship between the chemical repertory of an organism and its biology. Protein sequence profiles are very successful in capturing the main structural and functional features of a protein family, and can be used to assign new members to it based on matching of their sequences against these profiles. In this work, we extend this idea to chemical compounds, constructing a profile-inspired model for a set of related metabolites (those in the same biological pathway), based on a fragment-based vectorial representation of their chemical structures. RESULTS: We use this representation to predict the biological pathway of a chemical compound with good overall accuracy (AUC 0.74-0.90 depending on the database tested), and analyzed some factors that affect performance. The approach, which is compared with equivalent methods, can in addition detect those molecular fragments characteristic of a pathway. CONCLUSIONS: The method is available as a graphical interactive web server http://csbg.cnb.csic.es/iFragMent .


Asunto(s)
Proteínas , Programas Informáticos , Secuencia de Aminoácidos , Bases de Datos Factuales , Internet
2.
Int J Mol Sci ; 22(9)2021 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-34068603

RESUMEN

Genomic and phylogenetic analyses of various invertebrate phyla revealed the existence of genes that are evolutionarily related to the vertebrate's decapeptide gonadotropin-releasing hormone (GnRH) and the GnRH receptor genes. Upon the characterization of these gene products, encoding peptides and putative receptors, GnRH-related peptides and their G-protein coupled receptors have been identified. These include the adipokinetic hormone (AKH) and corazonin (CRZ) in insects and their cognate receptors that pair to form bioactive signaling systems, which network with additional neurotransmitters/hormones (e.g., octopamine and ecdysone). Multiple studies in the past 30 years have identified many aspects of the biology of these peptides that are similar in size to GnRH and function as neurohormones. This review briefly describes the main activities of these two neurohormones and their receptors in the fruit fly Drosophila melanogaster. The similarities and differences between Drosophila AKH/CRZ and mammalian GnRH signaling systems are discussed. Of note, while GnRH has a key role in reproduction, AKH and CRZ show pleiotropic activities in the adult fly, primarily in metabolism and stress responses. From a protein evolution standpoint, the GnRH/AKH/CRZ family nicely demonstrates the developmental process of neuropeptide signaling systems emerging from a putative common ancestor and leading to divergent activities in distal phyla.


Asunto(s)
Evolución Molecular , Hormonas de Insectos/genética , Proteínas de Insectos/genética , Neuropéptidos/genética , Neurotransmisores/genética , Oligopéptidos/genética , Ácido Pirrolidona Carboxílico/análogos & derivados , Secuencia de Aminoácidos/genética , Animales , Drosophila melanogaster/genética , Hormona Liberadora de Gonadotropina , Humanos , Filogenia , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética
3.
Nat Commun ; 12(1): 3431, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34103499

RESUMEN

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Asunto(s)
Genética Inversa , SARS-CoV-2/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , Culicidae/virología , Furina/metabolismo , Genoma Viral , Células HEK293 , Humanos , Ratones , Mutación/genética , Células 3T3 NIH , Reacción en Cadena de la Polimerasa , Células RAW 264.7 , Receptores Virales/metabolismo , Células Vero , Proteínas Virales/química , Replicación Viral
4.
Nat Commun ; 12(1): 3433, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34103506

RESUMEN

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.


Asunto(s)
Proteínas de la Envoltura de Coronavirus/química , Proteínas de la Envoltura de Coronavirus/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Secuencia de Aminoácidos , Proteínas de la Envoltura de Coronavirus/ultraestructura , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , SARS-CoV-2/fisiología , Homología Estructural de Proteína , Relación Estructura-Actividad
5.
Nat Commun ; 12(1): 3427, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34103518

RESUMEN

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.


Asunto(s)
Péptidos/química , Péptidos/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Determinación de Punto Final , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Oléicos/química , Péptidos/farmacología , Placebos , Conformación Proteica , Espectroscopía de Protones por Resonancia Magnética , Termodinámica , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología
6.
BMC Bioinformatics ; 22(1): 317, 2021 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-34112081

RESUMEN

BACKGROUND: To assign structural and functional annotations to the ever increasing amount of sequenced proteins, the main approach relies on sequence-based homology search methods, e.g. BLAST or the current state-of-the-art methods based on profile Hidden Markov Models, which rely on significant alignments of query sequences to annotated proteins or protein families. While powerful, these approaches do not take coevolution between residues into account. Taking advantage of recent advances in the field of contact prediction, we propose here to represent proteins by Potts models, which model direct couplings between positions in addition to positional composition, and to compare proteins by aligning these models. Due to non-local dependencies, the problem of aligning Potts models is hard and remains the main computational bottleneck for their use. METHODS: We introduce here an Integer Linear Programming formulation of the problem and PPalign, a program based on this formulation, to compute the optimal pairwise alignment of Potts models representing proteins in tractable time. The approach is assessed with respect to a non-redundant set of reference pairwise sequence alignments from SISYPHUS benchmark which have lowest sequence identity (between [Formula: see text] and [Formula: see text]) and enable to build reliable Potts models for each sequence to be aligned. This experimentation confirms that Potts models can be aligned in reasonable time ([Formula: see text] in average on these alignments). The contribution of couplings is evaluated in comparison with HHalign and independent-site PPalign. Although Potts models were not fully optimized for alignment purposes and simple gap scores were used, PPalign yields a better mean [Formula: see text] score and finds significantly better alignments than HHalign and PPalign without couplings in some cases. CONCLUSIONS: These results show that pairwise couplings from protein Potts models can be used to improve the alignment of remotely related protein sequences in tractable time. Our experimentation suggests yet that new research on the inference of Potts models is now needed to make them more comparable and suitable for homology search. We think that PPalign's guaranteed optimality will be a powerful asset to perform unbiased investigations in this direction.


Asunto(s)
Algoritmos , Proteínas , Secuencia de Aminoácidos , Humanos , Proteínas/genética , Alineación de Secuencia , Homología de Secuencia
7.
Int J Mol Sci ; 22(10)2021 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-34066037

RESUMEN

The multiple functions of the wild type Huntington's disease protein of the sea urchin Hemicentrotus pulcherrimus (Hp-Htt) have been examined using the anti-Hp-Htt antibody (Ab) raised against synthetic oligopeptides. According to immunoblotting, Hp-Htt was detected as a single band at around the 350 kDa region at the swimming blastula stage to the prism larva stage. From the 2-arm pluteus stage (2aPL), however, an additional smaller band at the 165 kDa region appeared. Immunohistochemically, Hp-Htt was detected in the nuclei and the nearby cytoplasm of the ectodermal cells from the swimming blastula stage, and the blastocoelar cells from the mid-gastrula stage. The Ab-positive signal was converged to the ciliary band-associated strand (CBAS). There, it was accompanied by several CBAS-marker proteins in the cytoplasm, such as glutamate decarboxylase. Application of Hp-Htt morpholino (Hp-Htt-MO) has resulted in shortened larval arms, accompanied by decreased 5-bromo-2-deoxyuridin (BrdU) incorporation by the ectodermal cells of the larval arms. Hp-Htt-MO also resulted in lowered ciliary beating activity, accompanied by a disordered swirling pattern formation around the body. These Hp-Htt-MO-induced deficiencies took place after the onset of CBAS system formation at the larval arms. Thus, Hp-Htt is involved in cell proliferation and the ciliary beating pattern regulation signaling system in pluteus larvae.


Asunto(s)
Cilios/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteína Huntingtina/metabolismo , Larva/fisiología , Erizos de Mar/fisiología , Natación , Secuencia de Aminoácidos , Animales , Proteína Huntingtina/genética , Homología de Secuencia
8.
Int J Mol Sci ; 22(10)2021 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-34066237

RESUMEN

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.


Asunto(s)
Amiloide/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Agregado de Proteínas , Conformación Proteica , Salmonella enterica/genética , Salmonella enterica/crecimiento & desarrollo , Homología de Secuencia
9.
Int J Mol Sci ; 22(10)2021 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-34068417

RESUMEN

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.


Asunto(s)
Ataxia/patología , Canales de Calcio/genética , Mutación , Adulto , Secuencia de Aminoácidos , Ataxia/congénito , Ataxia/etiología , Ataxia/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Niño , Femenino , Humanos , Masculino , Neuroimagen , Fenotipo , Conformación Proteica , Homología de Secuencia , Relación Estructura-Actividad , Adulto Joven
10.
Int J Mol Sci ; 22(10)2021 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-34069068

RESUMEN

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.


Asunto(s)
Citrus/crecimiento & desarrollo , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Citrus/genética , Citrus/metabolismo , Flores/genética , Flores/metabolismo , Proteínas de Dominio MADS/genética , Fenotipo , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Homología de Secuencia
11.
Anal Chim Acta ; 1169: 338627, 2021 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-34088371

RESUMEN

This study aimed to isolate FV-antibodies with biotin-binding activity from a FV-antibody library that was successfully screened on the outer membrane of E. coli. The aims were achieved by (1) preparing a library of FV-antibodies on the outer membrane of E. coli using autodisplay technology, (2) screening the FV-antibodies with biotin-binding activity from the FV-antibody library, and (3) synthesizing peptides (molecular weight of several kDa) from the biotin-binding amino acid sequence of FV-antibodies. An FV-antibody library with a diversity of 1.7 × 105 clones was prepared on the outer membrane of E. coli, using a surface display method called autodisplay technology. For the screening of biotin-binding FV-antibodies, the fluorescence-labeled biotin was introduced into the library, and the target E. coli with biotin-binding activity were screened using flow cytometry. For the screened E. coli clones, the binding affinity (KD) of Fv-antibodies against biotin was calculated and the binding properties of the screened FV-antibody were analyzed through competition assay with a synthetic peptide having the biotin-like activity. From the FRET experiment with the synthetic peptide corresponding to the CDR3 region of the screened Fv-antibody, the biotin-binding activity of the screened FV-antibody was proved to be originated from the CDR3. Finally, the applicability of the biotin-binding domain was demonstrated through the co-expression with a protein called Z-domain with antibody binding activity.


Asunto(s)
Biotina , Escherichia coli , Anticuerpos de Cadena Única/biosíntesis , Secuencia de Aminoácidos , Biotina/metabolismo , Escherichia coli/genética , Biblioteca de Genes , Biblioteca de Péptidos
12.
Klin Lab Diagn ; 66(6): 358-363, 2021 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-34105912

RESUMEN

Histatins are the most significant antimicrobial peptides (AMP) of saliva and there are 12 types of such AMP. Histatin molecules contain relatively high percent of histidine and tyrosine residues. This property allows to use well known from organic chemistry Pauly reaction for detection of protein bounded histidine and tyrosine residues (BHT), which are in fact characterize the summary content of all histatins in saliva. Aim of the present study was comparison of BHT with antimicrobial activity of salivary AMP fraction in patients with inflammatory diseases of upper airways (IDUA). Group of examined persons include 28 patients with different diagnoses: chronic pharyngitis (n=11), chronic tonsillitis (n=7), nasopharyngitis (n=5), pollinosis (n=5). Degree of intensity of inflammatory symptoms was estimated in balls. The algorithm of BHT analysis include following steps: freezing - thawing of saliva; removal of microparticles by centrifugation; separation of fraction lower than100 kDa; dialysis for free amino acids removal; Pauly reaction carrying out. Antimicrobial activities of saliva and its low molecular fractions were estimated towards Candida albicans cells by the spectrophotometric method with bromocresol purpur. Analysis of saliva sediments for coccoid microbiota was carried out by PCR method. Pauly reaction for histatins estimation in saliva of IDUA patients use here for the first time. The histatins levels (BHT) were significantly correlated with the intensity of inflammatory symptoms (r=0,975) and activity of low molecular salivary fraction (AMP activity) (r=0,824). The AMP activity/ BHT ratio, i.e. antimicrobial activity of histatin unit, decreased together with growth of inflammatory symptoms intensity (r=-0,944). Any considerable differences in coccoid microbiota frequency of finding at different diagnoses were not detected. The S. aureus frequency of occurrence was connected neither with inflammatory symptoms intensity (r=0,118), nor with BHT concentration (r=0,318). However S. pyogenes and S. pneumoniae frequencies of occurrence demonstrated the invert correlation towards these indexes: (r=-0,627/-0,614) and (r=-0,827/-0,864). Probably at the exacerbation forms of IDUA the S. pyogenes and S. pneumoniae growth controlled by high levels of histatins.


Asunto(s)
Histatinas , Staphylococcus aureus , Secuencia de Aminoácidos , Candida albicans , Humanos , Saliva , Proteínas y Péptidos Salivales
13.
Biomolecules ; 11(5)2021 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-34067685

RESUMEN

Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum ß-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25-50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.


Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Antivirales/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/química , Candida albicans/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dimerización , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , SARS-CoV-2/efectos de los fármacos
14.
Viruses ; 13(5)2021 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-34067878

RESUMEN

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.


Asunto(s)
Enzima Convertidora de Angiotensina 2/química , COVID-19/virología , Polisacáridos/química , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/química , Secuencia de Aminoácidos , Sitios de Unión , Glicosilación , Interacciones Microbiota-Huesped , Humanos , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Acoplamiento Viral
15.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-34078893

RESUMEN

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Asunto(s)
Magnesio/química , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Magnesio/metabolismo , Metilación , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Caperuzas de ARN/química , Caperuzas de ARN/genética , ARN Viral/química , ARN Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimología , SARS-CoV-2/ultraestructura , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genética
16.
Nat Commun ; 12(1): 3292, 2021 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-34078910

RESUMEN

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.


Asunto(s)
Autofagia/genética , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Cadenas Pesadas de Miosina/genética , Quinasas Relacionadas con NIMA/genética , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Línea Celular , Cilios/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Riñón/citología , Riñón/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocardio/citología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Quinasas Relacionadas con NIMA/deficiencia , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal
17.
Nat Commun ; 12(1): 3316, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34083524

RESUMEN

The methylglyoxal-derived hydroimidazolone isomer, MGH-1, is an abundant advanced glycation end-product (AGE) associated with disease and age-related disorders. As AGE formation occurs spontaneously and without an enzyme, it remains unknown why certain sites on distinct proteins become modified with specific AGEs. Here, we use a combinatorial peptide library to determine the chemical features that favor MGH-1. When properly positioned, tyrosine is found to play an active mechanistic role that facilitates MGH-1 formation. This work offers mechanistic insight connecting multiple AGEs, including MGH-1 and carboxyethylarginine (CEA), and reconciles the role of negative charge in influencing glycation outcomes. Further, this study provides clear evidence that glycation outcomes can be influenced through long- or medium-range cooperative interactions. This work demonstrates that these chemical features also predictably template selective glycation on full-length protein targets expressed in mammalian cells. This information is vital for developing methods that control glycation in living cells and will enable the study of glycation as a functional post-translational modification.


Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Productos Finales de Glicación Avanzada/química , Glicosilación , Células HEK293 , Humanos , Imidazoles/química , Imidazoles/metabolismo , Isomerismo , Biblioteca de Péptidos , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteínas/genética , Piruvaldehído/análogos & derivados , Piruvaldehído/química , Piruvaldehído/metabolismo
18.
BMC Bioinformatics ; 22(1): 297, 2021 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-34078264

RESUMEN

BACKGROUND: Feature extraction of protein sequences is widely used in various research areas related to protein analysis, such as protein similarity analysis and prediction of protein functions or interactions. RESULTS: In this study, we introduce FEGS (Feature Extraction based on Graphical and Statistical features), a novel feature extraction model of protein sequences, by developing a new technique for graphical representation of protein sequences based on the physicochemical properties of amino acids and effectively employing the statistical features of protein sequences. By fusing the graphical and statistical features, FEGS transforms a protein sequence into a 578-dimensional numerical vector. When FEGS is applied to phylogenetic analysis on five protein sequence data sets, its performance is notably better than all of the other compared methods. CONCLUSION: The FEGS method is carefully designed, which is practically powerful for extracting features of protein sequences. The current version of FEGS is developed to be user-friendly and is expected to play a crucial role in the related studies of protein sequence analyses.


Asunto(s)
Proteínas , Análisis de Secuencia de Proteína , Algoritmos , Secuencia de Aminoácidos , Aminoácidos , Filogenia , Proteínas/genética
19.
Rev Bras Parasitol Vet ; 30(2): e000421, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34076044

RESUMEN

Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.


Asunto(s)
Anaplasma marginale , Anaplasmosis , Enfermedades de los Bovinos , Secuencia de Aminoácidos , Anaplasma marginale/genética , Animales , Brasil , Bovinos , Genómica , Filogenia
20.
Nat Commun ; 12(1): 3168, 2021 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-34039967

RESUMEN

The rapid increase in the number of proteins in sequence databases and the diversity of their functions challenge computational approaches for automated function prediction. Here, we introduce DeepFRI, a Graph Convolutional Network for predicting protein functions by leveraging sequence features extracted from a protein language model and protein structures. It outperforms current leading methods and sequence-based Convolutional Neural Networks and scales to the size of current sequence repositories. Augmenting the training set of experimental structures with homology models allows us to significantly expand the number of predictable functions. DeepFRI has significant de-noising capability, with only a minor drop in performance when experimental structures are replaced by protein models. Class activation mapping allows function predictions at an unprecedented resolution, allowing site-specific annotations at the residue-level in an automated manner. We show the utility and high performance of our method by annotating structures from the PDB and SWISS-MODEL, making several new confident function predictions. DeepFRI is available as a webserver at https://beta.deepfri.flatironinstitute.org/ .


Asunto(s)
Biología Computacional/métodos , Aprendizaje Profundo , Modelos Biológicos , Estructura Terciaria de Proteína , Proteínas/fisiología , Secuencia de Aminoácidos , Bases de Datos de Proteínas/estadística & datos numéricos , Conjuntos de Datos como Asunto , Modelos Moleculares , Proteínas/ultraestructura , Relación Estructura-Actividad
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