RESUMEN

HLA-DQA1*05:115 shows an alanine at position 59 not described previously.

Asunto(s)

Alanina , Alelos , Cadenas alfa de HLA-DQ , Humanos , Cadenas alfa de HLA-DQ/genética , Alanina/genética , Exones , Prueba de Histocompatibilidad , Sustitución de Aminoácidos , Dominios Proteicos

RESUMEN

The N121 site on the spike protein of SARS-CoV-2 is associated with heme and its metabolite, biliverdin, which can affect antibody binding. Both N121T and N121S substitutions have been observed in natural conditions and in a hamster model of dual infection with SARS-CoV-2 and Influenza A virus. Serum pseudotype neutralization assays against HIV-1 particles carrying wild-type, N121T, and N121S spikes with immune mouse and human sera revealed that N121T and N121S mutations had a greater impact on serum neutralization than biliverdin treatment. Although N121T and N121S substitutions are not currently major SARS-CoV-2 variants of concern, this study could provide fundamental information to prepare for potential future mutations at the N121 site of SARS-CoV-2.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pruebas de Neutralización , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Humanos , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Ratones , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Sustitución de Aminoácidos , Mutación

RESUMEN

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Asunto(s)

Citomegalovirus , Proteínas del Envoltorio Viral , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Citomegalovirus/inmunología , Humanos , Animales , Ratones , Microscopía por Crioelectrón , Conformación Proteica , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Internalización del Virus , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Estabilidad Proteica , Vacunas contra Citomegalovirus/inmunología , Sustitución de Aminoácidos , Modelos Moleculares

RESUMEN

The epidemic and outbreaks of influenza B Victoria lineage (Bv) during 2019-2022 led to an analysis of genetic, epitopes, charged amino acids and Bv outbreaks. Based on the National Influenza Surveillance Network (NISN), the Bv 72 strains isolated during 2019-2022 were selected by spatio-temporal sampling, then were sequenced. Using the Compare Means, Correlate and Cluster, the outbreak data were analyzed, including the single nucleotide variant (SNV), amino acid (AA), epitope, evolutionary rate (ER), Shannon entropy value (SV), charged amino acid and outbreak. With the emergence of COVID-19, the non-pharmaceutical interventions (NPIs) made Less distant transmission and only Bv outbreak. The 2021-2022 strains in the HA genes were located in the same subset, but were distinct from the 2019-2020 strains (P < 0.001). The codon G → A transition in nucleotide was in the highest ratio but the transversion of C → A and T → A made the most significant contribution to the outbreaks, while the increase in amino acid mutations characterized by polar, acidic and basic signatures played a key role in the Bv epidemic in 2021-2022. Both ER and SV were positively correlated in HA genes (R = 0.690) and NA genes (R = 0.711), respectively, however, the number of mutations in the HA genes was 1.59 times higher than that of the NA gene (2.15/1.36) from the beginning of 2020 to 2022. The positively selective sites 174, 199, 214 and 563 in HA genes and the sites 73 and 384 in NA genes were evolutionarily selected in the 2021-2022 influenza outbreaks. Overall, the prevalent factors related to 2021-2022 influenza outbreaks included epidemic timing, Tv, Ts, Tv/Ts, P137 (B → P), P148 (B → P), P199 (P → A), P212 (P → A), P214 (H → P) and P563 (B → P). The preference of amino acid mutations for charge/pH could influence the epidemic/outbreak trends of infectious diseases. Here was a good model of the evolution of infectious disease pathogens. This study, on account of further exploration of virology, genetics, bioinformatics and outbreak information, might facilitate further understanding of their deep interaction mechanisms in the spread of infectious diseases.

Asunto(s)

Brotes de Enfermedades , Evolución Molecular , Gripe Humana , Mutación , Polimorfismo de Nucleótido Simple , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Gripe Humana/genética , Virus de la Influenza B/genética , Aminoácidos/genética , Epítopos/genética , Filogenia , Sustitución de Aminoácidos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética

RESUMEN

The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient's hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.

Asunto(s)

Mutación con Ganancia de Función , Trombocitopenia , Proteínas de Unión al GTP rap , Humanos , Masculino , Sustitución de Aminoácidos , Trasplante de Células Madre Hematopoyéticas , Síndromes de Inmunodeficiencia/genética , Mutación Missense , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Trombocitopenia/genética , Trombocitopenia/patología , Recién Nacido , Lactante , Preescolar , Niño

RESUMEN

Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a ß-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys → Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins.

Asunto(s)

Cisteína , Factor 2 de Crecimiento de Fibroblastos , Interacciones Hidrofóbicas e Hidrofílicas , Estabilidad Proteica , Cisteína/química , Cisteína/genética , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Mutación , Modelos Moleculares , Cristalografía por Rayos X , Sustitución de Aminoácidos , Humanos , Termodinámica

RESUMEN

Sapovirus (SaV) infection is increasing worldwide. Herein, we provided evidence of a significant increase in SaV infection in Japan during 2010-2022, primarily due to the considerable (p = 0.0003) rise of the GI.1 genotype. Furthermore, we found that all major and minor SaV outbreaks in Japan, including the largest SaV outbreak in 2021-2022, were caused by the GI.1 genotype. Therefore, to get insight into the underlying molecular mechanism behind this rising trend of the SaV GI.1 type, we selected 15 SaV GI.1 outbreak strains for complete genome analysis through next-generation sequencing. Phylogenetically, our strains remained clustered in different branches in lineages I and II among the GI.1 genotype. We showed all amino acid (aa) substitutions in different open reading frames (ORFs) in these strains. Importantly, we have demonstrated that the strains involved in the largest SaV outbreak in Japan in 2021-2022 belonged to lineage II and possessed the third ORF. We have identified some unique aa mutations in these major outbreak strains in the NS1 and NS6-NS7 regions that are thought to be associated with viral pathogenicity, cell tropism, and epidemiological competence. Thus, in addition to enriching the database of SaV's complete sequences, this study provides insights into its important mutations.

Asunto(s)

Infecciones por Caliciviridae , Brotes de Enfermedades , Evolución Molecular , Genoma Viral , Genotipo , Sistemas de Lectura Abierta , Filogenia , Sapovirus , Sapovirus/genética , Sapovirus/clasificación , Sapovirus/aislamiento & purificación , Humanos , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Japón/epidemiología , Genoma Viral/genética , Sistemas de Lectura Abierta/genética , Gastroenteritis/virología , Gastroenteritis/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Sustitución de Aminoácidos , Epidemiología Molecular , Secuenciación Completa del Genoma , Mutación

RESUMEN

The hepatitis B virus surface antigen's (HBsAg) 'a' determinant comprises a sequence of amino acid residues located in the major hydrophilic region of the S protein, whose exchanges are closely associated with compromising the antigenicity and immunogenicity of that antigen. The HBsAg is generally present in the bloodstream of individuals with acute or chronic hepatitis B virus (HBV) infection. It is classically known as the HBV infection marker, and is therefore the first marker to be investigated in the laboratory in the clinical hypothesis of infection by this agent. One of the factors that compromises the HBsAg detection in the bloodstream by the assays adopted in serological screening in both clinical contexts is the loss of S protein antigenicity. This can occur due to mutations that emerge in the HBV genome regions that encode the S protein, especially for its immunodominant region - the 'a' determinant. These mutations can induce exchanges of amino acid residues in the S protein's primary structure, altering its tertiary structure and the antigenic conformation, which may not be recognized by anti-HBs antibodies, compromising the infection diagnosis. In addition, these exchanges can render ineffective the anti-HBs antibodies action acquired by vaccination, compromise the effectiveness of the chronically HBV infected patient's treatment, and also the HBsAg immunogenicity, by promoting its retention within the cell. In this review, the residues exchange that alter the S protein's structure is revisited, as well as the mechanisms that lead to the HBsAg antigenicity loss, and the clinical, laboratory and epidemiological consequences of this phenomenon.

Asunto(s)

Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/química , Humanos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis B/virología , Hepatitis B/inmunología , Mutación , Sustitución de Aminoácidos , Anticuerpos contra la Hepatitis B/inmunología

RESUMEN

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new emerging coronavirus that caused coronavirus disease 2019 (COVID-19). Whole-genome tracking of SARS-CoV-2 enhanced our understanding of the mechanism of the disease, control, and prevention of COVID-19. METHODS: we analyzed 3368 SARS-CoV-2 protein sequences from Iran and compared them with 15.6 million global sequences in the GISAID database, using the Wuhan-Hu-1 strain as a reference. RESULTS: Our investigation revealed that NSP12-P323L, ORF9c-G50N, NSP14-I42V, membrane-A63T, Q19E, and NSP3-G489S were found to be the most frequent mutations among Iranian SARS-CoV-2 sequences. Furthermore, it was observed that more than 94% of the SARS-CoV-2 genome, including NSP7, NSP8, NSP9, NSP10, NSP11, and ORF8, had no mutations when compared to the Wuhan-Hu-1 strain. Finally, our data indicated that the ORF3a-T24I, NSP3-G489S, NSP5-P132H, NSP14-I42V, envelope-T9I, nucleocapsid-D3L, membrane-Q19E, and membrane-A63T mutations might be responsible factors for the surge in the SARS-CoV-2 Omicron variant wave in Iran. CONCLUSIONS: real-time genomic surveillance is crucial for detecting new SARS-CoV-2 variants, updating diagnostic tools, designing vaccines, and understanding adaptation to new environments.

Asunto(s)

COVID-19 , Genoma Viral , Mutación , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/clasificación , Irán/epidemiología , Humanos , COVID-19/virología , COVID-19/epidemiología , Sustitución de Aminoácidos , Glicoproteína de la Espiga del Coronavirus/genética

RESUMEN

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

Asunto(s)

ADN Nucleotidilexotransferasa , Simulación de Dinámica Molecular , Humanos , ADN Nucleotidilexotransferasa/metabolismo , ADN Nucleotidilexotransferasa/química , ADN Nucleotidilexotransferasa/genética , Especificidad por Sustrato , Desoxirribonucleótidos/metabolismo , Desoxirribonucleótidos/química , Nucleótidos de Timina/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiadenina/química , Enlace de Hidrógeno , Nucleótidos de Desoxiguanina/metabolismo , Nucleótidos de Desoxiguanina/química , Sustitución de Aminoácidos

RESUMEN

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in 2021 as a variant with heavy amino acid mutations in the spike protein, which is targeted by most vaccines, compared to previous variants. Amino acid substitutions in the spike proteins may alter their affinity for host viral receptors and the host interactome. Here, we found that the receptor-binding domain (RBD) of the omicron variant of SARS-CoV-2 exhibited an increased affinity for human angiotensin-converting enzyme 2, a viral cell receptor, compared to the prototype RBD. Moreover, we identified ß- and γ-actin as omicron-specific binding partners of RBD. Protein complex predictions revealed that many omicron-specific amino acid substitutions affected the affinity between RBD of the omicron variant and actin. Our findings indicate that proteins localized to different cellular compartments exhibit strong binding to the omicron RBD.

Asunto(s)

Actinas , Enzima Convertidora de Angiotensina 2 , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Actinas/metabolismo , COVID-19/virología , COVID-19/metabolismo , Dominios Proteicos , Mutación/genética , Sustitución de Aminoácidos , Receptores Virales/metabolismo , Receptores Virales/química , Sitios de Unión

RESUMEN

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Asunto(s)

Citocromos c , Simulación de Dinámica Molecular , Sitios de Unión , Ligandos , Citocromos c/metabolismo , Citocromos c/química , Citocromos c/genética , Peroxidasa/metabolismo , Peroxidasa/química , Peroxidasa/genética , Sustitución de Aminoácidos , Unión Proteica , Cianuros/metabolismo , Cianuros/química , Animales , Hemo/metabolismo , Hemo/química , Mutación

RESUMEN

Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1's pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the ß3-loop (S77D), ß3'-ß3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the ß3-loop and D103T in the ß3'-ß3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.

Asunto(s)

Hialuronoglucosaminidasa , Humanos , Sustitución de Aminoácidos , Dominio Catalítico , Moléculas de Adhesión Celular , Ácido Glutámico/metabolismo , Ácido Glutámico/química , Ácido Hialurónico/metabolismo , Ácido Hialurónico/química , Hialuronoglucosaminidasa/química , Hialuronoglucosaminidasa/metabolismo , Hialuronoglucosaminidasa/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación

RESUMEN

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models.

Asunto(s)

Modelos Genéticos , Filogenia , Archaea/genética , Funciones de Verosimilitud , Sustitución de Aminoácidos , Evolución Molecular , Eucariontes/genética

RESUMEN

The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) → Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.

Asunto(s)

Cobre , Chaperonas Moleculares , Superóxido Dismutasa-1 , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/química , Cobre/metabolismo , Zinc/metabolismo , Modelos Moleculares , Sustitución de Aminoácidos , Sitios de Unión , Oxidación-Reducción

RESUMEN

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The x-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4% and 7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

Asunto(s)

Prealbúmina , Prealbúmina/química , Prealbúmina/genética , Prealbúmina/metabolismo , Humanos , Modelos Moleculares , Cristalografía por Rayos X , Conformación Proteica , Multimerización de Proteína , Agregado de Proteínas , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Sitios de Unión , Sustitución de Aminoácidos

RESUMEN

A novel KPC variant, KPC-84, identified in a Klebsiella pneumoniae isolate from China, exhibits a threonine (T) to proline (P) amino acid substitution at Ambler position 243(T243P), altering from the KPC-2 sequence. Cloning and expression of blaKPC-84 in Escherichia coli, with subsequent MIC assessments, revealed increased resistance to ceftazidime-avibactam and significantly reduced carbapenemase activity compared to KPC-2. Kinetic measurements showed that KPC-84 exhibited sligthly higher hydrolysis of ceftazidime and reduced affinity for avibactam compared to KPC-2. This study emphasizes the emerging diversity of KPC variants with ceftazidime-avibactam resistance, underscoring the complexity of addressing carbapenem-resistant Klebsiella pneumoniae infections.

Asunto(s)

Antibacterianos , Compuestos de Azabiciclo , Proteínas Bacterianas , Ceftazidima , Combinación de Medicamentos , Infecciones por Klebsiella , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , beta-Lactamasas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Compuestos de Azabiciclo/farmacología , Ceftazidima/farmacología , Humanos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/tratamiento farmacológico , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , China , Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos

RESUMEN

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.

Asunto(s)

Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Recombinación Genética , Glicoproteína de la Espiga del Coronavirus , Enfermedades de los Porcinos , Animales , Secuencia de Aminoácidos , Sustitución de Aminoácidos , China/epidemiología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/epidemiología , Diarrea/virología , Diarrea/veterinaria , Diarrea/epidemiología , Variación Genética , Genotipo , Filogenia , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/clasificación , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/epidemiología

RESUMEN

BACKGROUND: De novo amino acid substitutions (DNS) frequently emerge among immunocompromised patients with chronic SARS-CoV-2 infection. While previous studies have reported these DNS, their significance has not been systematically studied. METHODS: We performed a review of DNS that emerged during chronic SARS-CoV-2 infection. We searched PubMed until June 2023 using the keywords "(SARS-CoV-2 or COVID-19) and (mutation or sequencing) and ((prolonged infection) or (chronic infection) or (long term))". We included patients with chronic SARS-CoV-2 infection who had SARS-CoV-2 sequencing performed for at least 3 time points over at least 60 days. We also included 4 additional SARS-CoV-2 patients with chronic infection of our hospital not reported previously. We determined recurrent DNS that has appeared in multiple patients and determined the significance of these mutations among epidemiologically-significant variants. FINDINGS: A total of 34 cases were analyzed, including 30 that were published previously and 4 from our hospital. Twenty two DNS appeared in ≥3 patients, with 14 (64%) belonging to lineage-defining mutations (LDMs) of epidemiologically-significant variants and 10 (45%) emerging among chronically-infected patients before the appearance of the corresponding variant. Notably, nsp9-T35I substitution (Orf1a T4175I) emerged in all three patients with BA.2.2 infection in 2022 before the appearance of Variants of Interest that carry nsp9-T35I as LDM (EG.5 and BA.2.86/JN.1). Structural analysis suggests that nsp9-T35I substitution may affect nsp9-nsp12 interaction, which could be critical for the function of the replication and transcription complex. INTERPRETATION: DNS that emerges recurrently in different chronically-infected patients may be used as a marker for potential epidemiologically-significant variants. FUNDING: Theme-Based Research Scheme [T11/709/21-N] of the Research Grants Council (See acknowledgements for full list).

Asunto(s)

Sustitución de Aminoácidos , COVID-19 , SARS-CoV-2 , Humanos , COVID-19/genética , COVID-19/virología , COVID-19/epidemiología , SARS-CoV-2/genética , Enfermedad Crónica , Mutación , Femenino , Masculino , Persona de Mediana Edad , Anciano

RESUMEN

Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage.

Asunto(s)

Inmunidad Innata , Proteínas no Estructurales Virales , Replicación Viral , Virus Zika , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/inmunología , Virus Zika/genética , Virus Zika/inmunología , Virus Zika/fisiología , Humanos , Infección por el Virus Zika/virología , Infección por el Virus Zika/inmunología , Animales , Chlorocebus aethiops , Células Vero , África Occidental , Sustitución de Aminoácidos , Línea Celular