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1.
Biochem Genet ; 60(6): 2299-2312, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35334059

RESUMEN

Viruses are the most common and abundant organisms in the marine environment. To better understand how cetaceans have adapted to this virus-rich environment, we compared cetacean virus-responsive genes to those from terrestrial mammals. We identified virus-responsive gene sequences in seven species of cetaceans, which we compared with orthologous sequences in seven terrestrial mammals. As a result of evolution analysis using the branch model and the branch-site model, 21 genes were selected using at least one model. IFN-ε, an antiviral cytokine expressed at mucous membranes, and its receptor IFNAR1 contain cetacean-specific amino acid substitutions that might change the interaction between the two proteins and lead to regulation of the immune system against viruses. Cetacean-specific amino acid substitutions in IL-6, IL-27, and the signal transducer and activator of transcription (STAT)1 are also predicted to alter the mucosal immune response of cetaceans. Since mucosal membranes are the first line of defense against the external environment and are involved in immune tolerance, our analysis of cetacean virus-responsive genes suggests that genes with cetacean-specific mutations in mucosal immunity-related genes play an important role in the protection and/or regulation of immune responses against viruses.


Asunto(s)
Cetáceos , Inmunidad Mucosa , Animales , Inmunidad Mucosa/genética , Filogenia , Cetáceos/genética , Mamíferos , Adaptación Fisiológica
2.
Angew Chem Int Ed Engl ; 60(36): 19766-19773, 2021 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-33963654

RESUMEN

Systematic inactivation of nonribosomal peptide synthetase (NRPS) domains and translocation of the thioesterase (TE) domain revealed several unprecedented nonlinear NRPS assembly processes during the biosynthesis of the cyclodepsipeptide WS9326A in Streptomyces sp. SNM55. First, two sets of type ΙΙ TE (TEΙΙ)-like enzymes mediate the shuttling of activated amino acids between two sets of stand-alone adenylation (A)-thiolation (T) didomain modules and an "A-less" condensation (C)-T module with distinctive specificities and flexibilities. This was confirmed by the elucidation of the affinities of the A-T didomains for the TEΙΙs and its structure. Second, the C-T didomain module operates iteratively and independently from other modules in the same protein to catalyze two chain elongation cycles. Third, this biosynthetic pathway includes the first example of module skipping, where the interpolated C and T domains are required for chain transfer.


Asunto(s)
Depsipéptidos/biosíntesis , Péptido Sintasas/metabolismo , Depsipéptidos/química , Estructura Molecular , Streptomyces/química , Streptomyces/metabolismo
3.
Curr Opin Infect Dis ; 33(2): 166-172, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32022744

RESUMEN

PURPOSE OF REVIEW: This review describes the major developments in the rationale for treating latent tuberculosis infection; new approaches to identifying persons with latent infection who are most likely to progress to active disease; and the development of novel short-course regimens for treatment of latent tuberculosis. RECENT FINDINGS: As many as one-third of the world's population has latent infection with Mycobacterium tuberculosis. Models demonstrate that tuberculosis will not be eliminated without large-scale treatment of persons with latent TB. Current tools for identifying persons at risk for active tuberculosis disease include TST and IGRA, which have poor positive predictive values. Newer approaches using gene expression profiling show promise and are being studied in the ongoing trials. Development of short-course regimens are a major advance in treatment of latent TB. Three months of rifapentine with isoniazid, 4 months of rifampin, and 1 month of rifapentine with isoniazid have been found to be noninferior to the standard 9 months of isoniazid. SUMMARY: Progress towards TB elimination can be accelerated by instituting public health measures that take into account new developments in identifying and treating persons with latent tuberculosis infection who are most likely to progress to active disease.


Asunto(s)
Antituberculosos/uso terapéutico , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/tratamiento farmacológico , Antituberculosos/administración & dosificación , Quimioterapia Combinada , Humanos , Factores de Riesgo
4.
Arch Biochem Biophys ; 693: 108570, 2020 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-32888908

RESUMEN

The hydrolysis of ß-lactam antibiotics by class C ß-lactamases proceeds through the acylation and the rate-determining deacylation steps mediated by the nucleophilic serine and the deacylation water, respectively. The pose of poor substrates such as carbapenems in the acylated enzyme is responsible for the low efficient deacylation reaction. Here we present the crystal structures of the Y150F variant of the ACC-1 class C ß-lactamase in the apo and acylated states. In the acylated enzyme complexed with two carbapenems, imipenem and meropenem, the lactam carbonyl oxygen is located in the oxyanion hole. However, the five-membered pyrroline ring displays a novel orientation that has not been reported so far. The ring is rotated such that its C3 carboxylate makes salt bridges with Lys67 and Ly315, which is accompanied by the side-chain rotamer change of Phe150. The C3 carboxylate is placed where the deacylation water occupies in the apo-enzyme, which, together with the displacement of the catalytic base residue at position 150, explains why carbapenems are poor substrates of ACC-1.


Asunto(s)
Carbapenémicos/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/efectos de los fármacos , Catálisis , Cristalografía por Rayos X , Conformación Proteica , beta-Lactamasas/química
5.
Artículo en Inglés | MEDLINE | ID: mdl-31451494

RESUMEN

ACC-1 is a plasmid-encoded class C ß-lactamase identified in clinical isolates of Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli ACC-1-producing bacteria are susceptible to cefoxitin, whereas they are resistant to oxyimino cephalosporins. Here, we depict crystal structures of apo ACC-1, adenylylated ACC-1, and acylated ACC-1 complexed with cefotaxime and cefoxitin. ACC-1 has noteworthy structural alterations in the R2 loop, the Ω loop, and the Phe119 loop located along the active-site rim. The adenylate covalently bonded to the nucleophilic serine reveals a tetrahedral phosphorus mimicking the deacylation transition state. Cefotaxime in ACC-1 has a proper conformation for the substrate-assisted catalysis in that its C-4 carboxylate and N-5 nitrogen are adequately located to facilitate the deacylation reaction. In contrast, cefoxitin in ACC-1 has a distinct conformation, in which those functional groups cannot contribute to catalysis. Furthermore, the orientation of the deacylating water relative to the acyl carbonyl group in ACC-1 is unfavorable for nucleophilic attack.


Asunto(s)
Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Catálisis , Cefotaxima/farmacología , Cefoxitina/farmacología , Cefalosporinas/farmacología , Pruebas de Sensibilidad Microbiana , Nitrógeno/química , Plásmidos/genética , beta-Lactamasas/química , beta-Lactamasas/genética
6.
PLoS One ; 19(9): e0307499, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39236042

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
7.
J Fungi (Basel) ; 9(2)2023 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-36836258

RESUMEN

Survival factor A (SvfA) in Aspergillus nidulans plays multiple roles in growth and developmental processes. It is a candidate for a novel VeA-dependent protein involved in sexual development. VeA is a key developmental regulator in Aspergillus species that can interact with other velvet-family proteins and enter into the nucleus to function as a transcription factor. In yeast and fungi, SvfA-homologous proteins are required for survival under oxidative and cold-stress conditions. To assess the role of SvfA in virulence in A. nidulans, cell wall components, biofilm formation, and protease activity were evaluated in a svfA-gene-deletion or an AfsvfA-overexpressing strain. The svfA-deletion strain showed decreased production of ß-1,3-glucan in conidia, a cell wall pathogen-associated molecular pattern, with a decrease in gene expression for chitin synthases and ß-1,3-glucan synthase. The ability to form biofilms and produce proteases was reduced in the svfA-deletion strain. We hypothesized that the svfA-deletion strain was less virulent than the wild-type strain; therefore, we performed in vitro phagocytosis assays using alveolar macrophages and analyzed in vivo survival using two vertebrate animal models. While phagocytosis was reduced in mouse alveolar macrophages challenged with conidia from the svfA-deletion strain, the killing rate showed a significant increase with increased extracellular signal-regulated kinase ERK activation. The svfA-deletion conidia infection reduced host mortality in both T-cell-deficient zebrafish and chronic granulomatous disease mouse models. Taken together, these results indicate that SvfA plays a significant role in the pathogenicity of A. nidulans.

8.
Sci Rep ; 13(1): 1005, 2023 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-36653390

RESUMEN

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.


Asunto(s)
Calefacción , Enfermedad del Hígado Graso no Alcohólico , Humanos , Animales , Ratones , Hígado/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Dieta Alta en Grasa , Ratones Endogámicos C57BL
9.
J Microbiol ; 60(11): 1086-1094, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36318359

RESUMEN

Fibroblast growth factor 11 (FGF11) is one of intracrine FGFs (iFGFs), which function within cells. Unlike canonical FGFs, FGF11 remains intracellularly and plays biological roles in FGF receptor (FGFR)-independent manner. Here, we established an expression system of recombinant FGF11 proteins in E. coli and investigated whether the extracellular administration of FGF11 can activate cellular signaling. Human FGF11 has two isoforms, FGF11a and FGF11b, depending on the presence of nuclear localization sequences (NLSs) in the N-terminus. Because these two isoforms are unstable, we prepared an FGF11a-Mut by substituting three cysteine residues in the NLS with serine and FGF11b-ΔC with C-terminal truncation. The introduction of mutation in the NLS improved the solubility of FGF11 prepared from E. coli. Exogenous addition of FGF11b and FGF11b-ΔC to BALB3T3 increased cell proliferation, while FGF11a-Mut exerted no effect. FGF11b-ΔC showed higher cell proliferation activity and FGFR signaling than FGF11b. The cell-proliferating activities of FGF11b and FGF11b-ΔC were blocked by an FGFR1 inhibitor or a recombinant FGFR1, confirming the FGFR1-dependent extracellular activity of FGF11b. The analysis of circular dichroism suggested that the C-terminus of FGF11 has an α-helical structure, which may affect its interaction with FGFR1. These results suggest that the N-and C-terminus of recombinant FGF11 are involved in the activation of FGFR1. The above results provide novel insights into the function and mechanism of FGF11 that may aid the development of useful ligands for FGFR regulation.


Asunto(s)
Escherichia coli , Factores de Crecimiento de Fibroblastos , Humanos , Escherichia coli/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Proliferación Celular , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo
10.
Nat Commun ; 12(1): 5931, 2021 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-34635673

RESUMEN

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.


Asunto(s)
Aurora Quinasa B/genética , Proteínas de Ciclo Celular/genética , Segregación Cromosómica , Mitosis , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Transactivadores/genética , Ácido Aspártico/metabolismo , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas Nucleares/deficiencia , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina/metabolismo , Transactivadores/deficiencia , Quinasa Tipo Polo 1
11.
PLoS One ; 13(10): e0204657, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30356254

RESUMEN

BACKGROUND AND AIM: Universal smoking cessation strategies are not always successful for minorities, among whom smoking is highly prevalent despite high intention to quit. This study identifies facilitators for smoking cessation, as perceived by minority male smokers, that can inform a culturally appropriate national plan for smoking prevention and cessation. METHODS: We conducted in 2013 a three-stage study among Arab minority male current and former smokers (ages 18-64) in Israel, among whom smoking is very high: first, a Concept Mapping (CM) study with 102 and 202 participants in the brainstorming, and sorting and rating phases respectively. Second, we assigned clusters identified in the CM study to contingency levels using the Behavioral Ecological Model (BEM). Third, we classified clusters into intervention functions and policies using the Behavior Change Wheel (BCW). FINDINGS: The CM study revealed 58 barriers and facilitators for smoking prevention and cessation that were sorted into 11 clusters by the participants. These clusters were analogous to four BEM level contingency of smoking (social, institutional, community and individual). We classified it into two main policy categories, based on the BCW: 1- restructuring the socio-political environment of smoking through affirmative government's policies towards Arab minority in Israel, and 2-developing a culturally appropriate plan for smoking cessation in Arab local authorities including: raising awareness about tobacco hazards; enforcing anti-smoking laws; strengthening community institutional action; providing smoking cessation services; considering raising prices for tobacco products, addressing psychological sources of smoking in Arab men. CONCLUSIONS: Our study revealed barriers, facilitators and contingencies of smoking prevention and cessation with two main policy action items among the Arab minority in Israel: changing the socio-political environment of smoking, and developing a culturally appropriate smoking prevention and cessation national plan. Our study framework can inform policies and culturally appropriate interventions for smoking prevention and cessation in other minorities.


Asunto(s)
Conductas Relacionadas con la Salud/fisiología , Grupos Minoritarios/psicología , Fumadores/psicología , Cese del Hábito de Fumar/psicología , Adolescente , Adulto , Árabes/psicología , Humanos , Israel , Masculino , Persona de Mediana Edad , Prevención del Hábito de Fumar/métodos , Productos de Tabaco/efectos adversos , Adulto Joven
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