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1.
Dis Markers ; 2022: 1118195, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36438904

RESUMO

Background: Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA. Methods: HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each). Results: In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1 µg/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression. Conclusions: In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Fator Xa/metabolismo , SARS-CoV-2 , Células Endoteliais/metabolismo , Subunidades Proteicas/metabolismo , Rivaroxabana/farmacologia , Rivaroxabana/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Mitocôndrias/metabolismo
2.
Nat Commun ; 13(1): 5989, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36220811

RESUMO

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.


Assuntos
ATPases Mitocondriais Próton-Translocadoras , Animais , Lipídeos , Mamíferos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Subunidades Proteicas/metabolismo , Prótons , Água
3.
Int J Mol Sci ; 23(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36232404

RESUMO

Spelt wheat (Triticum spelta L., 2n=6x=42, AABBDD) is a valuable source of new gene resources for wheat genetic improvement. In the present study, two novel high molecular weight glutenin subunits (HMW-GS) 1Ax2.1* at Glu-A1 and 1By19* at Glu-B1 from German spelt wheat were identified. The encoding genes of both subunits were amplified and cloned by allele-specific PCR (AS-PCR), and the complete sequences of open reading frames (ORF) were obtained. 1Ax2.1* with 2478 bp and 1By19* with 2163 bp encoded 824 and 720 amino acid residues, respectively. Molecular characterization showed that both subunits had a longer repetitive region, and high percentage of α-helices at the N- and C-termini, which are beneficial for forming superior gluten macropolymers. Protein modelling by AlphaFold2 revealed similar three-diamensional (3D) structure features of 1Ax2.1* with two x-type superior quality subunits (1Ax1 and 1Ax2*) and 1By19* with four y-type superior quality subunits (1By16, 1By9, 1By8 and 1By18). Four cysteine residues in the three x-type subunits (1Ax2.1*, 1Ax1 and 1Ax2*) and the cysteine in intermediate repeat region of y-type subunits were not expected to participate in intramolecular disulfide bond formation, but these cysteines might form intermolecular disulfide bonds with other glutenins and gliadins to enhance gluten macropolymer formation. The SNP-based molecular markers for 1Ax2.1* and 1By19* genes were developed, which were verified in different F2 populations and recombination inbred lines (RILs) derived from crossing between spelt wheat and bread wheat cultivars. This study provides data on new glutenin genes and molecular markers for wheat quality improvement.


Assuntos
Cisteína , Triticum , Cisteína/metabolismo , Dissulfetos/metabolismo , Glutens/química , Peso Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Triticum/genética , Triticum/metabolismo
4.
Anal Chem ; 94(41): 14410-14418, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36206384

RESUMO

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.


Assuntos
Nucleotídeos de Guanina , Proteínas Heterotriméricas de Ligação ao GTP , Polarização de Fluorescência , Nucleotídeos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Ligação Proteica , Subunidades Proteicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
5.
Nature ; 611(7935): 399-404, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289347

RESUMO

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.


Assuntos
Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase , Complexos Multienzimáticos , Animais , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/ultraestrutura , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Aminoácidos , Glucose , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo
6.
Anticancer Drugs ; 33(10): 1004-1011, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36255067

RESUMO

GNG5 is suggested to exert a critical effect on tumor development in human beings; however, its function and related mechanism within breast cancer (BC) are still unclear. In this regard, the present work focused on identifying and evaluating GNG5's function and revealing its possible molecular mechanism. Subcutaneous tumorigenesis model of nude mice and in-vitro cell model was established. The relationship between GNG5 expression and BC was studied through knockdown and overexpression experiments. The proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of liver cancer cell lines overexpressing or silencing GNG5 were detected. Furthermore, the pathway mechanism of GNG5 was evaluated at the molecular level and was performed to further verify the possible targets and mechanisms of action. In comparison with that in normal tissue, GNG5 level within BC tissue was higher. In addition, GNG5 overexpression stimulated BC cell proliferation, invasion, migration and EMT. BC cells with reduced GNG5 expression exhibited significant decreases in glucose uptake, lactate levels, and ATP concentrations. In addition, GNG5 knockdown inhibited Wnt/ß-catenin signaling. This study indicates that GNG5 may generate a vital function in BC. The results of the current work demonstrated GNG5's effect on BC pathological process, also providing a reference for developing new targeted therapies for BC.


Assuntos
Neoplasias da Mama , Subunidades gama da Proteína de Ligação ao GTP , Via de Sinalização Wnt , Animais , Feminino , Humanos , Camundongos , Trifosfato de Adenosina/metabolismo , beta Catenina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Glucose , Glicólise , Lactatos/farmacologia , Camundongos Nus , Subunidades Proteicas/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo
7.
Nat Commun ; 13(1): 5824, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36192412

RESUMO

The photochemical reaction center (RC) features a dimeric architecture for charge separation across the membrane. In green sulfur bacteria (GSB), the trimeric Fenna-Matthews-Olson (FMO) complex mediates the transfer of light energy from the chlorosome antenna complex to the RC. Here we determine the structure of the photosynthetic supercomplex from the GSB Chlorobaculum tepidum using single-particle cryogenic electron microscopy (cryo-EM) and identify the cytochrome c subunit (PscC), two accessory protein subunits (PscE and PscF), a second FMO trimeric complex, and a linker pigment between FMO and the RC core. The protein subunits that are assembled with the symmetric RC core generate an asymmetric photosynthetic supercomplex. One linker bacteriochlorophyll (BChl) is located in one of the two FMO-PscA interfaces, leading to differential efficiencies of the two energy transfer branches. The two FMO trimeric complexes establish two different binding interfaces with the RC cytoplasmic surface, driven by the associated accessory subunits. This structure of the GSB photosynthetic supercomplex provides mechanistic insight into the light excitation energy transfer routes and a possible evolutionary transition intermediate of the bacterial photosynthetic supercomplex from the primitive homodimeric RC.


Assuntos
Chlorobi , Proteínas de Bactérias/metabolismo , Bacterioclorofilas , Chlorobi/metabolismo , Citocromos c/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Subunidades Proteicas/metabolismo
8.
Zhonghua Gan Zang Bing Za Zhi ; 30(9): 954-961, 2022 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-36299189

RESUMO

Objective: To analyze guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) expression based on bioinformatics, so as to evaluate its role and its relationship with survival rate during the occurrence and development of hepatocellular carcinoma. Methods: GEPIA, UALCAN and HPA databases were used to analyze the expression level of GNB2L1 and its relationship with HCC survival rate. Mutations in the GNB2L1 gene and their impact on survival were analyzed using the cBioPortal database. LinkedOmics database was used to analyze GNB2L1-related genes in HCC. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed simultaneously. STEING database was used to construct the GNB2L1 protein interaction network. TIMER database was used to analyze the relationship between GNB2L1 gene expression and immune infiltration in hepatocellular carcinoma. Differential expression of GNB2L1 in plasma platelets of HCC patients and healthy controls was analyzed using mRNA-based sequencing technology. Data between groups were compared using an independent-samples t-test. Results: GNB2L1 expression level was significantly increased in HCC tissues (P<0.05), and its expression was significantly correlated with body weight, classification and stage (P<0.05). The overall survival rate was higher in GNB2L1 low expression group (P<0.001). GNB2L1 and its related genes were related to biological process regulation, metabolic process, protein binding, oxidative phosphorylation, JAK-STAT signaling pathway, Ras signaling pathway and so on. GNB2L1 had interaction with RPS12, RPS11 and RPL19, and participated in multiple biological processes such as liver regeneration and positive regulation of endogenous apoptotic signaling pathway. GNB2L1 expression was significantly positively correlated with the infiltration degree of various immune cells in HCC (P<0.05). Cox regression analysis showed that GNB2L1 was an independent risk factor for lower survival rate in patients with HCC [Hazard ratio (95% confidence interval)=1.456 (1.034~2.051), P=0.031]. GNB2L1expression levels were significantly higher in platelets of HCC patients than that of healthy controls (10.40±1.36 vs. 9.58±0.51, t=2.194, P=0.037). Conclusion: GNB2L1 has high expression and close relationship to survival rate in HCC. Therefore, GNB2L1 may be a potential biomarker of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Biologia Computacional , Neoplasias Hepáticas/patologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro , Nucleotídeos de Guanina , Expressão Gênica , Biomarcadores Tumorais/genética
9.
Int J Mol Sci ; 23(19)2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36232696

RESUMO

The NMDA receptor is a Ca2+-permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs contain two GluN2 subunits, of which 2A and 2B are predominant in the forebrain. Moreover, the relative contribution of 2A vs. 2B is controlled both developmentally and in an activity-dependent manner. The GluN2 subtype influences the biophysical properties of the receptor through difference in their N-terminal extracellular domain and transmembrane regions, but they also have large cytoplasmic Carboxyl (C)-terminal domains (CTDs) which have diverged substantially during evolution. While the CTD identity does not influence NMDAR subunit specific channel properties, it determines the nature of CTD-associated signalling molecules and has been implicated in mediating the control of subunit composition (2A vs. 2B) at the synapse. Historically, much of the research into the differential function of GluN2 CTDs has been conducted in vitro by over-expressing mutant subunits, but more recently, the generation of knock-in (KI) mouse models have allowed CTD function to be probed in vivo and in ex vivo systems without heterologous expression of GluN2 mutants. In some instances, findings involving KI mice have been in disagreement with models that were proposed based on earlier approaches. This review will examine the current research with the aim of addressing these controversies and how methodology may contribute to differences between studies. We will also discuss the outstanding questions regarding the role of GluN2 CTD sequences in regulating NMDAR subunit composition, as well as their relevance to neurodegenerative disease and neurodevelopmental disorders.


Assuntos
Doenças Neurodegenerativas , Transtornos do Neurodesenvolvimento , Receptores de N-Metil-D-Aspartato , Animais , Modelos Animais de Doenças , Crescimento e Desenvolvimento/genética , Crescimento e Desenvolvimento/fisiologia , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/fisiopatologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Sinapses/genética , Sinapses/metabolismo , Sinapses/fisiologia
10.
Biochemistry (Mosc) ; 87(10): 1109-1118, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36273879

RESUMO

In photosynthetic reaction centers of intact photosystem I (PSI) complexes from cyanobacteria, electron transfer at room temperature occurs along two symmetrical branches of redox cofactors A and B at a ratio of ~3 : 1 in favor of branch A. Previously, this has been indirectly demonstrated using pulsed absorption spectroscopy and more directly by measuring the decay modulation frequencies of electron spin echo signals (electron spin echo envelope modulation, ESEEM), which allows to determine the distance between the separated charges of the primary electron donor P700+ and phylloquinone acceptors A1A- and A1B- in the symmetric redox cofactors branches A and B. In the present work, these distances were determined using ESEEM in PSI complexes lacking three 4Fe-4S clusters, FX, FA, and FB, and the PsaC protein subunit (the so-called P700-A1 core), in which phylloquinone molecules A1A and A1B serve as the terminal electron acceptors. It was shown that in the P700-A1 core preparations, the average distance between the centers of the P700+A1- ion-radical pair at a temperature of 150 K in an aqueous glycerol solution and in a dried trehalose matrix, as well as in a trehalose matrix at 280 K, is ~25.5 Å, which corresponds to the symmetrical electron transfer along the A and B branches of redox cofactors at a ratio of 1 : 1. Possible reasons for the change in the electron transfer symmetry in PSI upon removal of the PsaC subunit and 4Fe-4S clusters FX, FA, and FB are discussed.


Assuntos
Proteínas Ferro-Enxofre , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Ferro/metabolismo , Elétrons , Vitamina K 1 , Trealose , Subunidades Proteicas/metabolismo , Glicerol , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Enxofre/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Cinética
11.
Nat Plants ; 8(10): 1191-1201, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36229605

RESUMO

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI-light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.


Assuntos
Cianobactérias , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Plastocianina , Complexos de Proteínas Captadores de Luz/metabolismo , Subunidades Proteicas/metabolismo , Cianobactérias/metabolismo , Água/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo
12.
Nutrients ; 14(18)2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36145058

RESUMO

The outermost constituent of many bacterial cells is represented by an S-layer, i.e., a semiporous lattice-like layer composed of self-assembling protein subunits called S-layer proteins (Slps). These proteins are involved in several processes, such as protecting against environmental stresses, mediating bacterial adhesion to host cells, and modulating gut immune response. Slps may also act as a scaffold for the external display of additional cell surface proteins also named S-layer associated proteins (SLAPs). Levilactobacillus brevis is an S-layer forming lactic acid bacterium present in many different environments, such as sourdough, milk, cheese, and the intestinal tract of humans and animals. This microorganism exhibits probiotic features including the inhibition of bacterial infection and the improvement of human immune function. The potential role of Slps in its probiotic and biotechnological features was documented. A shotgun proteomic approach was applied to identify in a single experiment both the Slps and the SLAPs pattern of five different L. brevis strains isolated from traditional sourdoughs of the Southern Italian region. This study reveals that these closely related strains expressed a specific pattern of surface proteins, possibly affecting their peculiar properties.


Assuntos
Lactobacillus brevis , Proteômica , Animais , Humanos , Ácido Láctico/metabolismo , Lactobacillus brevis/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Subunidades Proteicas/metabolismo
13.
Int J Mol Sci ; 23(18)2022 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-36142449

RESUMO

The cytochrome bc1 complex is an essential component of the mitochondrial respiratory chain of the yeast Saccharomyces cerevisiae. It is composed of ten protein subunits, three of them playing an important role in electron transfer and proton pumping across the inner mitochondrial membrane. Cytochrome b, the central component of this respiratory complex, is encoded by the mitochondrial genome, whereas all the other subunits are of nuclear origin. The assembly of all these subunits into the mature and functional cytochrome bc1 complex is therefore a complicated process which requires the participation of several chaperone proteins. It has been found that the assembly process of the mitochondrial bc1 complex proceeds through the formation of distinct sub-complexes in an ordered sequence. Most of these sub-complexes have been thoroughly characterized, and their molecular compositions have also been defined. This study critically analyses the results obtained so far and highlights new possible areas of investigation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Citocromos b/genética , Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Subunidades Proteicas/metabolismo , Prótons , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Theriogenology ; 193: 1-10, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36115287

RESUMO

Copper is widely used as a feeding additive to promote livestock growth. However, excessive copper can be excreted with feces, causing heavy metal pollution and aggravating environmental problems. At the same time, studies have found that excess copper can cause damage to reproductive function and reduce gamete quality. Here, we explored the effects of adding different concentrations of copper to the culture medium on porcine oocytes. First polar body extrusion rate, embryo development, and intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) ΔΨm, adenosine triphosphate(ATP) content, and acetylation of lysine 9 on histone H3 protein subunit (H3K9ac) were assessed. Results demonstrated that Cu exposure causes abnormalities in mitochondrial function and epigenetic modification, resulting in increased oxidative stress and levels of ROS, ultimately leading to a decreased porcine oocyte quality. In addition, we found melatonin can protect porcine oocytes from those damages. Notably, Nrf2 protein expression was significantly increased by copper exposure, meanwhile, Nrf2 signaling pathway inhibitor ML385 significantly attenuated the protective role of melatonin on oxidative stress induced by copper exposure. In summary, our study demonstrates that copper activates the Nrf2 pathway and impairs oocyte maturation by inducing oxidative stress, leading to poor quality of porcine oocytes, and the changes can be reversed by melatonin.


Assuntos
Melatonina , Trifosfato de Adenosina/metabolismo , Animais , Cobre/toxicidade , Histonas/metabolismo , Lisina/metabolismo , Melatonina/metabolismo , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oócitos/fisiologia , Estresse Oxidativo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Suínos
15.
Nature ; 610(7931): 394-401, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36171293

RESUMO

Filoviruses, including Ebola virus, pose an increasing threat to the public health. Although two therapeutic monoclonal antibodies have been approved to treat the Ebola virus disease1,2, there are no approved broadly reactive drugs to control diverse filovirus infection. Filovirus has a large polymerase (L) protein and the cofactor viral protein 35 (VP35), which constitute the basic functional unit responsible for virus genome RNA synthesis3. Owing to its conservation, the L-VP35 polymerase complex is a promising target for broadly reactive antiviral drugs. Here we determined the structure of Ebola virus L protein in complex with tetrameric VP35 using cryo-electron microscopy (state 1). Structural analysis revealed that Ebola virus L possesses a filovirus-specific insertion element that is essential for RNA synthesis, and that VP35 interacts extensively with the N-terminal region of L by three protomers of the VP35 tetramer. Notably, we captured the complex structure in a second conformation with the unambiguous priming loop and supporting helix away from polymerase active site (state 2). Moreover, we demonstrated that the century-old drug suramin could inhibit the activity of the Ebola virus polymerase in an enzymatic assay. The structure of the L-VP35-suramin complex reveals that suramin can bind at the highly conserved NTP entry channel to prevent substrates from entering the active site. These findings reveal the mechanism of Ebola virus replication and may guide the development of more powerful anti-filovirus drugs.


Assuntos
Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Ebolavirus , Proteínas Virais Reguladoras e Acessórias , Antivirais/farmacologia , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Ebolavirus/enzimologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Viral/biossíntese , Suramina/química , Suramina/metabolismo , Suramina/farmacologia , Suramina/uso terapêutico , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/ultraestrutura , Replicação Viral
16.
Proc Natl Acad Sci U S A ; 119(39): e2202485119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122241

RESUMO

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.


Assuntos
Opsinas dos Cones , GTP Fosfo-Hidrolases , Fosfatos , Proteínas RGS , Células Fotorreceptoras Retinianas Cones , Catálise , Opsinas dos Cones/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Osmose , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas RGS/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Vitamina A/metabolismo
17.
Sci Rep ; 12(1): 16236, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36171252

RESUMO

The SARS-CoV-2 prefusion spike protein is characterized by a high degree of flexibility and temporal transformations associated with its multifunctional behavior. In this study, we have examined the dynamics of the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein in detail. Its primary, binding subdomain with human Angiotensin Covering Enzyme II includes a highly conspicuous flap or loop that is part of a beta hairpin loop structural motif. Dynamic details of the RBD obtained through RMSF and Order Parameter calculations are consistent with structural details including the stability of "glue" points or dominant interaction energy residues of the RBD in the Up and Down states with its neighboring N-terminal domain (NTD) protomer. The RBD flap in the Up state protomer periodically obstructs the binding site on an approximate 70 ns time interval and is reminiscent of an HIV-1 protease polypeptide flap that opens and closes to modulate that enzymes activity. No claim is made here regarding the possible modulating role of the flap; however, the flap may be a potential site for therapeutic targeting aimed at keeping it in the closed state, as previously demonstrated in the inhibition of the HIV-1 protease polypeptide. The RBD primary binding subdomain is further shown to have not only similar dynamics but, also, an approximate 30% sequence similarity to the HIV-1 protease polypeptide.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2 , Angiotensinas/metabolismo , Protease de HIV , Humanos , Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo
18.
Subcell Biochem ; 99: 351-377, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36151382

RESUMO

Photosystem I (PSI) is a protein complex functioning in light-induced charge separation, electron transfer, and reduction reactions of ferredoxin in photosynthesis, which finally results in the reduction of NAD(P)- to NAD(P)H required for the fixation of carbon dioxide. In eukaryotic algae, PSI is associated with light-harvesting complex I (LHCI) subunits, forming a PSI-LHCI supercomplex. LHCI harvests and transfers light energy to the PSI core, where charge separation and electron transfer reactions occur. During the course of evolution, the number and sequences of protein subunits and the pigments they bind in LHCI change dramatically depending on the species of organisms, which is a result of adaptation of organisms to various light environments. In this chapter, I will describe the structure of various PSI-LHCI supercomplexes from different organisms solved so far either by X-ray crystallography or by cryo-electron microscopy, with emphasis on the differences in the number, structures, and association patterns of LHCI subunits associated with the PSI core found in different organisms.


Assuntos
Complexos de Proteínas Captadores de Luz , Complexo de Proteína do Fotossistema I , Dióxido de Carbono/metabolismo , Microscopia Crioeletrônica , Ferredoxinas/metabolismo , Complexos de Proteínas Captadores de Luz/química , NAD/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Subunidades Proteicas/metabolismo
19.
Subcell Biochem ; 99: 317-350, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36151381

RESUMO

Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.


Assuntos
Mamíferos , Receptores de Ácido Caínico , Animais , Mamíferos/metabolismo , Mutação , Subunidades Proteicas/metabolismo , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo
20.
J Gen Physiol ; 154(10)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-35939271

RESUMO

The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ ßENaC ≫ αENaC in both organs. Transcript abundance in the kidney, measured with digital-drop PCR and RNAseq, was similar for the three subunits. In both organs, the calculated protein expression of all subunits was much larger than that required to account for maximal Na+ currents measured in these cells, implying a large excess of subunit protein. Whole-kidney biotinylation indicated that at least 5% of ß and γ subunits in the kidney and 3% in the colon were expressed on the surface under conditions of salt restriction, which maximizes ENaC-dependent Na+ transport. This indicates a 10- to 100-fold excess of ßENaC and γENaC subunits at the surface relative to the requirement for channel activity. We conclude that these epithelia make much more ENaC protein than is required for the physiological function of the channel. This could facilitate rapid regulation of the channels at the cell surface by insuring a large population of inactive, recruitable subunits.


Assuntos
Canais Epiteliais de Sódio , Sódio , Animais , Membrana Celular/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Rim/metabolismo , Subunidades Proteicas/metabolismo , Ratos , Sódio/metabolismo
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