RESUMO
Ubiquitination is a representative post-translational modification that tags target proteins with ubiquitin to induce protein degradation or modify their functions. Deubiquitinating enzymes (DUBs) play a crucial role in reversing this process by removing ubiquitin from target proteins. Among them, USP2a has emerged as a promising target for cancer therapy due to its oncogenic properties in various cancer types, but its inhibitors have been limited. In this study, our aim was to optimize the structure of ML364, a USP2a inhibitor, and synthesize a series of its derivatives to develop potent USP2a inhibitors. Compound 8v emerged as a potential USP2a inhibitor with lower cytotoxicity compared to ML364. Cellular assays demonstrated that compound 8v effectively reduced the levels of USP2a substrates and attenuated cancer cell growth. We confirmed its direct interaction with the catalytic domain of USP2a and its selective inhibitory activity against USP2a over other USP subfamily proteins (USP7, 8, or 15). In conclusion, compound 8v has been identified as a potent USP2a inhibitor with substantial potential for cancer therapy.
Assuntos
Endopeptidases , Ubiquitina , Endopeptidases/química , Proteólise , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.
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Neoplasias , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , UbiquitinaçãoRESUMO
Biomolecular operations, which involve the conversion of molecular signals or interactions into specific functional outputs, are fundamental to the field of biology and serve as the important foundation for the design of diagnostic and therapeutic systems. To maximize their functionalities and broaden their applicability, it is crucial to develop novel outputs and facile chemical transformation methods. With this aim, in this study, we present a straightforward method for converting nucleic acid signals into fluorescein outputs that exhibit a wide range of functionalities. This operation is designed through a DNA-templated reaction based on riboflavin-photocatalyzed oxidation of dihydrofluorescein, which is readily prepared by simple NaBH4 reduction of the fluorescein with no complicated chemical caging steps. The templated photooxidation exhibits high efficiency (kapp = 2.7 × 10-3/s), generating a clear fluorescein output signal distinguishable from a low background, originating from the high stability of the synthesized dihydrofluorescein. This facile and efficient operation allows the nucleic acid-initiated activation of various fluorescein functions, such as fluorescence and artificial oxidase activity, which are applied in the design of novel bioanalytical systems, including fluorescent and colorimetric DNA sensors. The operation presented herein would expand the scope of biomolecular circuit systems for diagnostic and therapeutic applications.
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Ácidos Nucleicos , Fluoresceína , Colorimetria , CorantesRESUMO
This study investigated the characteristics of transmission routes of COVID-19 cluster infections (⩾10 linked cases within a short period) in Gangwon Province between 22 February 2020 and 31 May 2021. Transmission routes were divided into five major categories and 35 sub-categories according to the relationship between the infector and the infectee and the location of transmission. A total of 61 clusters occurred during the study period, including 1741 confirmed cases (55.7% of all confirmed cases (n = 3125)). The the five major routes of transmission were as follows: 'using (staying in) the same facility (50.7%), 'cohabiting family members' (23.3%), 'social gatherings with acquaintances' (10.8%), 'other transmission routes' (7.0%), and 'social gatherings with non-cohabiting family members/relatives' (5.5%). For transmission caused by using (staying in) the same facility, the highest number of confirmed cases was associated with churches, followed by medical institutions (inpatient), sports facilities, military bases, offices, nightlife businesses, schools, restaurants, day-care centres and kindergarten, and service businesses. Our analysis highlights specific locations with frequent transmission of infections, and transmission routes that should be targeted in situations where adherence to disease control rules is difficult.
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COVID-19/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/epidemiologia , COVID-19/mortalidade , Criança , Pré-Escolar , Análise por Conglomerados , Família , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Ocupações/estatística & dados numéricos , República da Coreia/epidemiologia , Fatores de Risco , Adulto JovemRESUMO
ABA is a phytohormone involved in diverse plant events such as seed germination and drought response. An F-box protein functions as a substrate receptor of the SCF complex and is responsible for ubiquitination of target proteins, triggering their subsequent degradation mediated by ubiquitin proteasome system. Here, we have isolated a gene named ARABIDOPSIS F-BOX PROTEIN HYPERSENSITIVE TO ABA 1 (AFA1) that was upregulated by ABA. AFA1 interacted with adaptor proteins of the SCF complex, implying its role as a substrate receptor of the complex. Its loss of function mutants, afa1 seedlings, exhibited ABA-hypersensitivity, including delayed germination in the presence of ABA. Moreover, loss of AFA1 led to increased drought tolerance in adult plants. Microarray data with ABA treatments indicated that 129 and 219 genes were upregulated or downregulated, respectively, by more than three times in afa1 relative to the wild type. Among the upregulated genes in afa1, the expression of 28.7% was induced by more than three times in the presence of ABA, while only 9.3% was repressed to the same extent. These data indicate that AFA1 is involved in the downregulation of many ABA-inducible genes, in accordance with the ABA-hypersensitive phenotype of afa1. Epistasis analysis showed that AFA1 could play a role upstream of ABI4 and ABI5 in the ABA signaling for germination inhibition. Collectively, our findings suggest that AFA1 is a novel F-box protein that negatively regulates ABA signaling.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Ácido Abscísico/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secas , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Sementes/metabolismoRESUMO
We investigated the protective effect and mechanisms of apigenin against cognitive impairments in a scopolamine-injected mouse model. Our results showed that intraperitoneal (i.p.) injection of scopolamine leads to learning and memory dysfunction, whereas the administration of apigenin (synthetic compound, 100 and 200 mg/kg/day) improved cognitive ability, which was confirmed by behavioral tests such as the T-maze test, novel objective recognition test, and Morris water maze test in mice. In addition, scopolamine-induced lipid peroxidation in the brain was attenuated by administration of apigenin. To further evaluate the protective mechanisms of apigenin on cognitive and memory function, Western blot analysis was carried out. Administration of apigenin decreased the B-cell lymphoma 2-associated X/B-cell lymphoma 2 (Bax/Bcl-2) ratio and suppressed caspase-3 and poly ADP ribose polymerase cleavage. Furthermore, apigenin down-regulated the ß-site amyloid precursor protein-cleaving enzyme, along with presenilin 1 (PS1) and PS2 protein levels. Apigenin-administered mice showed lower protein levels of a receptor for advanced glycation end-products, whereas insulin-degrading enzyme, brain-derived neurotrophic factor (BDNF), and tropomyosin receptor kinase B (TrkB) expression were promoted by treatment with apigenin. Therefore, this study demonstrated that apigenin is an active substance that can improve cognitive and memory functions by regulating apoptosis, amyloidogenesis, and BDNF/TrkB signaling pathways.
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Apigenina/farmacologia , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Neurônios/efeitos dos fármacos , Escopolamina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Modelos Animais de Doenças , Peroxidação de Lipídeos/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Glycine oxidase (GO) is an enzyme that catalyzes the oxidation of the primary and secondary amines of various chemicals, including glycine, and the enzyme has been applied in a variety of fields, such as biosensor and genetically modified glyphosate resistance plants. Here, we report that the gene product of BC0747 from Bacillus cereus (BcGO) shows oxidase activity for glycine and small d-amino acids, such as d-proline and d-alanine. We also determined the crystal structure of BcGO complexed with the FAD cofactor at a 2.36 Å resolution and revealed how the cofactor binds to the deep pocket of the enzyme. We performed the molecular docking calculation of the glycine substrate to the BcGO structure and identified how the carboxyl- and amine-groups of the d-amino acid are stabilized at the substrate binding site. Structural analysis of BcGO also provided information on the structural basis for the stereospecificity of the enzyme to d-amino acids. In addition, we placed the glyphosate molecule, a plant herbicide, at the substrate binding site, and explained how the mutation of Gly51 to arginine enhances enzyme activity.
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Aminoácido Oxirredutases/química , Aminoácidos/química , Bacillus cereus/enzimologia , Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Domínio Catalítico , Flavina-Adenina Dinucleotídeo/química , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Estereoisomerismo , GlifosatoRESUMO
Prunus cerasoides (PC) products contain relatively high levels of flavones and isoflavones and may be potential sources of phytoestrogens for postmenopausal symptom relief. We assessed the PC extract (PCE) and its representative constituents in vitro with assays for estrogen receptor alpha binding, estrogen response element transcriptional activity, cell proliferation, and gene expression changes for pS2 in MCF-7 cells. PCE and its compounds showed strong estrogen receptor binding affinities and estrogen response element induction. A previously undescribed compound (designated as compound 18), now identified as being gentisic acid, 5-O-ß-D-(6'-O-trans-4-coumaroyl)-glucopyranoside, also showed potent estrogenic properties and induced proliferation of MCF-7 cells. PCE was evaluated for its in vivo uterotrophic effects in immature female rats as well as for its lipid lowering effects in estrogen-deprived animals. For ovariectomized rats and aged female mice, PCE-treated groups had lower plasma triglyceride levels compared with control and, for the same comparison, had reduced serum levels of liver stress/damage markers. Our results point to strong estrogenic activities and beneficial metabolic effects for PCE, with properties that put PC and its extracts as promising sources of phytoestrogens for symptom relief in menopausal and postmenopausal cases.
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Estrogênios/uso terapêutico , Extratos Vegetais/química , Prunus/química , Animais , Modelos Animais de Doenças , Estrogênios/farmacologia , Feminino , Humanos , Células MCF-7/metabolismo , Camundongos , RoedoresRESUMO
The bone tissue is a dynamic complex that constitutes of several interdependent systems and is continuously remodeled through the concerted actions of bone cells. Osteoblasts are mononucleated cells, derived from mesenchymal stem cells, responsible for bone formation. Osteoclasts are large multinucleated cells that differentiate from hematopoietic progenitors of the myeloid lineage and are responsible for bone resorption. The lineage-specific differentiation of bone cells requires an epigenetic regulation of gene expressions involving chromatin dynamics. The key step for understanding gene regulatory networks during bone cell development lies in characterizing the chromatin modifying enzymes responsible for reorganizing and potentiating particular chromatin structure. This review covers the histone-modifying enzymes involved in bone development, discusses the impact of enzymes on gene expression, and provides future directions and clinical significance in this area.
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Remodelação Óssea , Diferenciação Celular , Código das Histonas , Animais , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , OsteogêneseRESUMO
Osteoporosis is a common disorder of bone remodeling, caused by the imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Recently, we reported that matrix metalloproteinase-9 (MMP-9)-dependent histone H3 proteolysis is a key event for proficient osteoclast formation. Although it has been reported that several MMP-9 inhibitors, such as tetracycline and its derivatives, show an inhibitory effect on osteoclastogenesis, the molecular mechanisms for this are not fully understood. Here we show that tetracycline analogs, especially tigecycline and minocycline, inhibit osteoclast formation by blocking MMP-9-mediated histone H3 tail cleavage. Our molecular docking approach found that tigecycline and minocycline are the most potent inhibitors of MMP-9. We also observed that both inhibitors significantly inhibited H3 tail cleavage by MMP-9 in vitro. These compounds inhibited receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast formation by blocking the NFATc1 signaling pathway. Furthermore, MMP-9-mediated H3 tail cleavage during osteoclast differentiation was selectively blocked by these compounds. Treatment with both tigecycline and minocycline rescued the osteoporotic phenotype induced by prednisolone in a zebrafish osteoporosis model. Our findings demonstrate that the tetracycline analogs suppress osteoclastogenesis via MMP-9-mediated H3 tail cleavage, and suggest that MMP-9 inhibition could offer a new strategy for the treatment of glucocorticoid-induced osteoporosis.
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Histonas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Minociclina/farmacologia , Osteogênese/efeitos dos fármacos , Tigeciclina/farmacologia , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Modelos Moleculares , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Peixe-ZebraRESUMO
Chitosan is a biocompatible natural polysaccharide, which has been employed as a polymeric scaffold for versatile, systemic delivery platforms and for locally injectable gels with temperature-sensitive viscosity modulation. Despite the extensive investigation on the chemical modification strategies, however, most of the chitosan-based delivery platforms have been focused on the encapsulation of hydrophobic drugs, which can be simply adsorbed on the chitosan scaffolds by hydrophobic interaction via the postparticle-formation drug-loading process. Herein, we present the facile formation of a cisplatin-coordinated chitosan nanoplatform by exploiting the divalent metal (PtII)-mediated conformational changes of chitosan chains, which allows for the simultaneous drug-loading and nanoparticle formation. To this end, the native chitosan has been chemically modified with short polyethylene glycol and malonic acid as a colloidal stabilizer and a bidentate chelating ligand for PtII coordination, respectively. The resulting PtII-modified polyampholytic chitosan (PtII-MPC) has been self-associated in aqueous media by hydrophobic segregation into a compact nanostructure, which exhibited an attenuated viscosity and pH-sensitive release of PtII compounds. Once the cationic drug molecules have been released under mild acidic conditions, the neutralized PtII-free MPC undergoes interchain flocculation near the isoelectric point because of the polyampholytic property, possibly allowing for the facilitated endosomal escape during the cellular endocytosis by the known membrane perturbation property of chitosan.
Assuntos
Nanopartículas , Quitosana , Cisplatino , Portadores de Fármacos , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , ViscosidadeRESUMO
The hPrp19-CDC5L complex plays a crucial role during human pre-mRNA splicing by catalytic activation of the spliceosome. In order to elucidate the molecular architecture of the hPrp19-CDC5L complex, the crystal structure of CTNNBL1, one of the major components of this complex, was determined. Unlike canonical ARM-repeat proteins such as ß-catenin and importin-α, CTNNBL1 was found to contain a twisted and extended ARM-repeat structure at the C-terminal domain and, more importantly, the protein formed a stable dimer. A highly negatively charged patch formed in the N-terminal ARM-repeat domain of CTNNBL1 provides a binding site for CDC5L, a binding partner of the protein in the hPrp19-CDC5L complex, and these two proteins form a complex with a stoichiometry of 2:2. These findings not only present the crystal structure of a novel ARM-repeat protein, CTNNBL1, but also provide insights into the detailed molecular architecture of the hPrp19-CDC5L complex.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ciclo Celular/química , Enzimas Reparadoras do DNA/química , Glicoproteínas de Membrana/química , Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Proteínas Reguladoras de Apoptose/genética , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Proteínas Nucleares/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Precursores de RNA/química , Precursores de RNA/genética , Splicing de RNA/genética , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Sequências de Repetição em TandemRESUMO
Crotonase from Clostridium acetobutylicum (CaCRT) is an enzyme that catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway. To investigate the molecular mechanism underlying n-butanol biosynthesis, we determined the crystal structures of the CaCRT protein in apo- and acetoacetyl-CoA bound forms. Similar to other canonical crotonase enzymes, CaCRT forms a hexamer by the dimerization of two trimers. A crystal structure of CaCRT in complex with acetoacetyl-CoA revealed that Ser69 and Ala24 to be signature residues of CaCRT, which results in a distinct ADP binding mode wherein the ADP moiety is bound at a different position compared with other crotonases. We also revealed that the substrate specificity of crotonase enzymes is determined by both the structural feature of the α3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed α3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. The key residues involved in substrate binding, enzyme catalysis and substrate specificity were confirmed by site-directed mutagenesis.
Assuntos
Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Clostridium acetobutylicum/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
BACKGROUND: Cyberbullying research has gained considerable attention among scholars due to the proliferation and diversification of the digital media platforms. Although cyberbullying research has a robust empirical nature, the research is more difficult to define and deserves close investigation. In response to the situation, we have examined topics, forms, context, media, predictive factors, outcomes, utilization of methodology, and research trends in cyberbullying research from 2014 to 2023. METHODS: This study analyzed 3,588 articles to investigate the trend and development of cyberbullying research using the Scopus academic databases. RESULTS: 85.87% of research focused on cyberbullying as 'social issues'. 71.57% of articles focused on cyberbullying itself rather than specific forms of cyberbullying. 50.81% of articles focused on cyberbullying in middle/high school contexts. Most research did not state any predictive factors (39.83%) or outcomes (60.17%) suggested. The predominant research method used in cyberbullying research was via surveys (30.69%), while the second phase of research dominated the cyberbullying research agenda (victims and bullies in cyberbullying). CONCLUSIONS: Although Cyberbullying research has increased, broadened, and diversified over time, it still focuses primarily on the first phase of the research agenda of cyberbullying issues. The findings of this study provides a framework for new insights for future cyberbullying research and practices by suggesting exploration to different mechanisms of cyberbullying such as: victims/bullies, effects, and improvement of cyberbullying beyond the issues of cyberbullying itself.
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This study aimed to identify the time to diagnosis among COVID-19 patients and factors associated with delayed diagnosis (DD). Data from COVID-19 patients in Gangwon, South Korea, diagnosed between 22 February 2020 and 29 January 2022, were analyzed, excluding asymptomatic cases and those who underwent mandatory testing. DD was defined as a period exceeding 2 or more days from symptom recognition to COVID-19 diagnosis. Univariate analysis was performed to investigate the demographic characteristics, COVID-19 symptoms, and underlying medical conditions associated with DD, followed by multivariate logistic regression analysis for significant variables. Among 2683 patients, 584 (21.8%) were diagnosed within a day of symptom onset. DD rates were lower in patients with febrile symptoms but higher among those with cough, myalgia, or anosmia/ageusia. High-risk underlying medical conditions were not significantly associated with DD. Older age groups, the Wonju medical service area, time of diagnosis between November 2020 and July 2021, symptom onset on nonworkdays, and individuals in nonwhite collar sectors were significantly associated with increased DD risks. These findings were consistent in the sensitivity analysis. This study underscores the need for enhanced promotion and system adjustments to ensure prompt testing upon symptom recognition.
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COVID-19 , Diagnóstico Tardio , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , República da Coreia/epidemiologia , Masculino , Feminino , Diagnóstico Tardio/estatística & dados numéricos , Pessoa de Meia-Idade , Adulto , Idoso , Adulto Jovem , Adolescente , Fatores de Risco , SARS-CoV-2 , Idoso de 80 Anos ou maisRESUMO
Introduction: Cartilage regeneration is a challenging issue due to poor regenerative properties of tissues. Electrospun nanofibers hold enormous potentials for treatments of cartilage defects. However, nanofibrous materials used for the treatment of cartilage defects often require physical and/or chemical modifications to promote the adhesion, proliferation, and differentiation of cells. Thus, it is highly desirable to improve their surface properties with functionality. We aim to design hydrophilic, adhesive, and compound K-loaded nanofibers for treatments of cartilage defects. Methods: Hydrophilic and adhesive compound K-containing polycaprolactone nanofibers (CK/PCL NFs) were prepared by coatings of gallic acid-conjugated chitosan (CHI-GA). Therapeutic effects of CHI-GA/CK/PCL NFs were assessed by the expression level of genes involved in the cartilage matrix degradation, inflammatory response, and lipid accumulations in the chondrocytes. In addition, Cartilage damage was evaluated by safranin O staining and immunohistochemistry of interleukin-1ß (IL-1ß) using OA animal models. To explore the pathway associated with therapeutic effects of CHI-GA/CK/PCL NFs, cell adhesion, phalloidin staining, and the expression level of integrins and peroxisome proliferator-activated receptor (PPARs) were evaluated. Results: CHI-GA-coated side of the PCL NFs showed hydrophilic and adhesive properties, whereas the unmodified opposite side remained hydrophobic. The expression levels of genes involved in the degradation of the cartilage matrix, inflammation, and lipogenesis were decreased in CHI-GA/CK/PCL NFs owing to the release of CK. In vivo implantation of CHI-GA/CK/PCL NFs into the cartilage reduced cartilage degradation induced by destabilization of the medial meniscus (DMM) surgery. Furthermore, the accumulation of lipid deposition and expression levels of IL-1ß was reduced through the upregulation of PPAR. Conclusion: CHI-GA/CK/PCL NFs were effective in the treatments of cartilage defects by inhibiting the expression levels of genes involved in cartilage degradation, inflammation, and lipogenesis as well as reducing lipid accumulation and the expression level of IL-1ß via increasing PPAR.
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Quitosana , Ginsenosídeos , Nanofibras , Animais , Receptores Ativados por Proliferador de Peroxissomo , Cartilagem , Inflamação/tratamento farmacológico , Regeneração , LipídeosRESUMO
The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.
Assuntos
Proteínas de Transporte , Ácido N-Acetilneuramínico , Proteínas de Membrana Transportadoras/genética , Conformação Molecular , DissulfetosRESUMO
OBJECTIVE: The impact of the government-initiated senior employment program (GSEP) on geriatric depressive symptoms is underexplored. Unearthing this connection could facilitate the planning of future senior employment programs and geriatric depression interventions. In the present study, we aimed to elucidate the possible association between geriatric depressive symptoms and GSEP in older adults. METHODS: This study employed data from 9,287 participants aged 65 or older, obtained from the 2020 Living Profiles of Older People Survey. We measured depressive symptoms using the Korean version of the 15-item Geriatric Depression Scale. The principal exposure of interest was employment status and GSEP involvement. Data analysis involved multiple linear regression. RESULTS: Employment, independent of income level, showed association with decreased depressive symptoms compared to unemployment (p<0.001). After adjustments for confounding variables, participation in GSEP jobs showed more significant reduction in depressive symptoms than non-GSEP jobs (ß=-0.968, 95% confidence interval [CI]=-1.197 to -0.739, p<0.001 for GSEP jobs, ß=-0.541, 95% CI=-0.681 to -0.401, p<0.001 for non-GSEP jobs). Notably, the lower income tertile in GSEP jobs showed a substantial reduction in depressive symptoms compared to all income tertiles in non-GSEP jobs. CONCLUSION: The lower-income GSEP group experienced lower depressive symptoms and life dissatisfaction compared to non-GSEP groups regardless of income. These findings may provide essential insights for the implementation of government policies and community-based interventions.
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Epilepsy, marked by abnormal and excessive brain neuronal activity, is linked to the activation of L-type voltage-gated calcium channels (LTCCs) in neuronal membranes. LTCCs facilitate the entry of calcium (Ca2+) and other metal ions, such as zinc (Zn2+) and magnesium (Mg2+), into the cytosol. This Ca2+ influx at the presynaptic terminal triggers the release of Zn2+ and glutamate to the postsynaptic terminal. Zn2+ is then transported to the postsynaptic neuron via LTCCs. The resulting Zn2+ accumulation in neurons significantly increases the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, contributing to reactive oxygen species (ROS) generation and neuronal death. Amlodipine (AML), typically used for hypertension and coronary artery disease, works by inhibiting LTCCs. We explored whether AML could mitigate Zn2+ translocation and accumulation in neurons, potentially offering protection against seizure-induced hippocampal neuronal death. We tested this by establishing a rat epilepsy model with pilocarpine and administering AML (10 mg/kg, orally, daily for 7 days) post-epilepsy onset. We assessed cognitive function through behavioral tests and conducted histological analyses for Zn2+ accumulation, oxidative stress, and neuronal death. Our findings show that AML's LTCC inhibition decreased excessive Zn2+ accumulation, reactive oxygen species (ROS) production, and hippocampal neuronal death following seizures. These results suggest amlodipine's potential as a therapeutic agent in seizure management and mitigating seizures' detrimental effects.
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Background: The brain-gut axis has emerged as a potential target in neurodegenerative diseases, including dementia, as individuals with dementia exhibit distinct gut microbiota compositions. Fecal microbiota transplantation (FMT), the transfer of fecal solution from a healthy donor to a patient, has shown promise in restoring homeostasis and cognitive enhancement. Objective: This study aimed to explore the effects of FMT on specific cognitive performance measures in Alzheimer's dementia (AD) patients and investigate the relationship between cognition and the gut microbiota by evaluating changes in gene expression following FMT. Methods: Five AD patients underwent FMT, and their cognitive function [Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB)] was assessed before and after FMT. The patients' fecal samples were analyzed with 16S rRNA to compare the composition of their gut microbiota. We also assessed modifications in the serum mRNA expression of patients' genes related to lipid metabolism using serum RNA sequencing and quantitative real-time polymerase chain reaction. Results: Significant improvements in cognitive function, as measured by the MMSE (pre- and post-FMT was 13.00 and 18.00) and MoCA were seen. The MoCA scores at 3 months post-FMT (21.0) were the highest (12.0). The CDR-SOB scores at pre- and post-FMT were 10.00 and 5.50, respectively. Analysis of the gut microbiome composition revealed changes via 16S rRNA sequencing with an increase in Bacteroidaceae and a decrease in Enterococcaceae. Gene expression analysis identified alterations in lipid metabolism-related genes after FMT. Conclusion: These findings suggest a link between alterations in the gut microbiome, gene expression related to lipid metabolism, and cognitive function. The study highlights the importance of gut microbiota in cognitive function and provides insights into potential biomarkers for cognitive decline progression. FMT could complement existing therapies and show potential as a therapeutic intervention to mitigate cognitive decline in AD.