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1.
Biochim Biophys Acta Gene Regul Mech ; 1862(10): 194442, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31634638

RESUMO

MyoD is a determining transcription factor involved in myogenic cell differentiation. Post translational modifications of MyoD, including phosphorylation and acetylation, can regulate its transcription activity. Inhibition of protein arginine methyltransferase 1 (PRMT1) leads to insufficient muscle differentiation. However, little is known about arginine methylation in regulating MyoD activity. Here, we demonstrated that MyoD interacts with PRMT1 via its bHLH domain. MyoD could be methylated by PRMT1 at R121. Moreover, R111 and R121 of MyoD are responsible for MyoD-mediated myogenin gene transcription in C2C12 cells. PRMT1 promotes MyoD-mediated myogenin expression, for which the enzymatic activity of PRMT1 is needed. The arginine methylation of MyoD by PRMT1 enhances its DNA binding activity and transactivation. Our data help to further clarify the molecular mechanism of PRMT1 in regulating muscle cell differentiation and provide a new therapeutic target for diseases caused by the abnormal differentiation of muscle cells.


Assuntos
Proteína MyoD/genética , Miogenina/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Transcrição Genética , Arginina/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Desenvolvimento Muscular/genética , Processamento de Proteína Pós-Traducional/genética
2.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615004

RESUMO

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.


Assuntos
ADP Ribose Transferases/química , Arginina/química , Peptídeos/química , Xanthomonas/química , ADP Ribose Transferases/genética , Sequência de Aminoácidos/genética , Arginina/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Oxigênio/química , Peptídeos/genética , Plantas/genética , Plantas/microbiologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais/genética , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
3.
Enzyme Microb Technol ; 131: 109424, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615672

RESUMO

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.


Assuntos
Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Animais , Arginina/genética , Estabilidade Enzimática , Vaga-Lumes/enzimologia , Cinética , Luciferases de Vaga-Lume/química , Lisina/genética , Proteínas Mutantes/química , Mutação Puntual , Conformação Proteica
4.
Molecules ; 24(17)2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466351

RESUMO

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.


Assuntos
DNA/metabolismo , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Substituição de Aminoácidos , Arginina/genética , Domínio Catalítico , Dano ao DNA , Histidina/genética , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fenilalanina/genética , Ligação Proteica , Uracila-DNA Glicosidase/genética
5.
Amino Acids ; 51(8): 1103-1127, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31267155

RESUMO

Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term "repression" was proposed by Vogel (The chemical basis of heredity, The John Hopkins Press, Baltimore, 1957) (in opposition to induction) to describe the relative decrease in acetylornithinase production in Escherichia coli cells upon arginine supplementation, whereas the term "regulon" was coined by Maas and Clark (J Mol Biol 8:365-370, 1964) for the ensemble of arginine biosynthetic genes dispersed over the E. coli chromosome but all subjected to regulation by the trans-acting argR gene product. Since then, unraveling of the molecular mechanisms controlling arginine biosynthesis, catabolism, and transport in and out the cell, have revealed moonlighting activities of enzymes and transcriptional regulators that generate unexpected interconnections between at first sight totally unrelated cellular processes, and have continued to replenish scientific knowledge and stimulated creative thinking. Furthermore, arginine is much more than just a common amino acid for protein synthesis. It may also be used as sole source of nitrogen by E. coli and a source of nitrogen, carbon and energy by many other bacteria. It is a substrate for the synthesis of polyamines, and important for the extreme acid resistance of E. coli. Furthermore, the guanidino group of arginine is well suited to engage in multiple interactions involving hydrogen bonds and ionic interactions with proteins and nucleic acids. Here, we combine major historical discoveries with current state of the art knowledge on arginine biosynthesis, catabolism and transport, and especially the regulation of these processes in E. coli, with reference to other microorganisms.


Assuntos
Arginina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Arginina/genética , Transporte Biológico , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Transcrição Genética
6.
Immunogenetics ; 71(7): 479-487, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270568

RESUMO

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.


Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Antígenos Heterófilos/genética , Arginina/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Mutagênese Sítio-Dirigida , Mutação Puntual , Suínos
7.
Mol Biol Evol ; 36(10): 2328-2339, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220870

RESUMO

Because of the degeneracy of the genetic code, multiple codons are translated into the same amino acid. Despite being "synonymous," these codons are not equally used. Selective pressures are thought to drive the choice among synonymous codons within a genome, while GC content, which is typically attributed to mutational drift, is the major determinant of variation across species. Here, we find that in addition to GC content, interspecies codon usage signatures can also be detected. More specifically, we show that a single amino acid, arginine, is the major contributor to codon usage bias differences across domains of life. We then exploit this finding and show that domain-specific codon bias signatures can be used to classify a given sequence into its corresponding domain of life with high accuracy. We then wondered whether the inclusion of codon usage codon autocorrelation patterns, which reflects the nonrandom distribution of codon occurrences throughout a transcript, might improve the classification performance of our algorithm. However, we find that autocorrelation patterns are not domain-specific, and surprisingly, are unrelated to tRNA reusage, in contrast to previous reports. Instead, our results suggest that codon autocorrelation patterns are a by-product of codon optimality throughout a sequence, where highly expressed genes display autocorrelated "optimal" codons, whereas lowly expressed genes display autocorrelated "nonoptimal" codons.


Assuntos
Archaea/genética , Bactérias/genética , Eucariotos/genética , Arginina/genética , Composição de Bases , Humanos , Anotação de Sequência Molecular , RNA de Transferência/metabolismo
8.
J Neurooncol ; 144(1): 79-87, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240524

RESUMO

PURPOSE: Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II-III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous affecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specific B and T cell reactivity in blood and tumor tissue of LGG patients. METHODS: Sera from IDH1R132H-mutated LGG patients (n = 27) were assayed for the presence of a neo-specific antibody response using ELISA. In addition, PBMCs (n = 36) and tumor-infiltrating lymphocytes (TILs, n = 10) were measured for T cell activation markers and IFN-γ production by flow cytometry and ELISA. In some assays, frequencies of CD4 T cells specific for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. RESULTS: Despite high sensitivity of our assay, we failed to detect IDH1R132H-specific IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following pre-enrichment of frequencies of IDH1R132H-specific CD4 T cells. Finally, we did not detect IDH1R132H-specific CD4 T cells among TILs. CONCLUSIONS: The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufficiently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients.


Assuntos
Arginina/genética , Linfócitos B/imunologia , Glioma/imunologia , Isocitrato Desidrogenase/genética , Mutação , Linfócitos T/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Glioma/genética , Glioma/patologia , Humanos , Prognóstico
9.
PLoS Pathog ; 15(6): e1007746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194856

RESUMO

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.


Assuntos
Arginina/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Arginina/genética , Transporte Biológico Ativo/genética , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Toxoplasmose/genética , Toxoplasmose/patologia
10.
Diabetes Res Clin Pract ; 152: 135-145, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31102685

RESUMO

INTRODUCTION: Many studies have evaluated the association of paraoxonase-1 (PON1) gene polymorphisms with enzyme activity and concentration in type 2 diabetes mellitus (T2DM). However, the exact impact of these polymorphisms is not still obvious. Hence, we conducted a systematic review and meta-analysis to clarify the association of PON1 polymorphisms with its enzyme characteristics in T2DM patients and non-diabetic individuals. METHODS: We searched electronic databases including PubMed, Web of Science, Embase and Scopus for publications by April 2018. The pooled response ratio (rr) for the association and their corresponding 95% confidence intervals (CIs) were calculated using a fixed-effect model. RESULTS: Fifteen relevant studies fulfilled our inclusion criteria. The results showed a 1.25-fold increase in total PON1 activity in non-diabetic group against T2DM patients (p-value = 0.024). Also, only Q192R and L55M polymorphisms had sufficient studies to be included in the meta-analysis. All three genotypes of Q192R polymorphism showed significantly different activities between the study groups with the highest pooled effect size for RR genotype (rrQQ < rrQR < rrRR) while this difference was seen only in LL genotype of L55M polymorphism. Therefore, Q192R polymorphism was more correlated with type 2 diabetes mellitus. In case of concentration, there was no significant differences between two groups (p-value = 0.897). CONCLUSION: Current meta-analysis suggested that the observed difference of total PON1 activity was due to the different activity of various genotypes of PON1 enzyme in case of L55M and Q192R polymorphisms so that LL and RR genotypes had the most important role in the establishment of mentioned difference.


Assuntos
Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Diabetes Mellitus Tipo 2/genética , Mutação com Ganho de Função , Polimorfismo Genético , Substituição de Aminoácidos , Arginina/genética , Diabetes Mellitus Tipo 2/metabolismo , Ativação Enzimática/genética , Feminino , Genótipo , Ácido Glutâmico/genética , Humanos , Leucina/genética , Masculino , Metionina/genética
11.
Mol Cell Biochem ; 458(1-2): 133-142, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31093850

RESUMO

Changes in the ecto-5'-nucleotidase activity-an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and L-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73-/- (CD73-/-) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73-/- mice age groups were characterized by reduced L-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73-/- mice lungs in comparison to EC isolated from WT lungs. The L-Arginine/ADMA ratio in the CD73-/- decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73-/- mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73-/- compared to controls. Blood (erythrocyte) Adenosine-5'-triphosphate and Adenosine-5'-diphosphate levels were reduced in favor to higher blood Adenosine-5'-monophosphate concentration in CD73-/- mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired L-arginine metabolism. CD73 activity seems to protect endothelium.


Assuntos
5'-Nucleotidase/deficiência , Arginina/sangue , Endotélio Vascular/metabolismo , Difosfato de Adenosina/sangue , Difosfato de Adenosina/genética , Trifosfato de Adenosina/sangue , Trifosfato de Adenosina/genética , Animais , Arginina/genética , Endotélio Vascular/patologia , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/sangue , Interleucina-6/genética , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo III/genética , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/genética
12.
Mol Cell ; 74(4): 713-728.e6, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30981631

RESUMO

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas Nucleares/genética , Sequências Repetitivas de Aminoácidos/genética , Esclerose Amiotrófica Lateral/patologia , Arginina/genética , Nucléolo Celular/química , Nucléolo Celular/genética , Dipeptídeos/genética , Humanos , Peptídeos/genética , Poli A/genética , RNA Ribossômico/genética
13.
Mol Cell ; 74(5): 922-935.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30979585

RESUMO

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.


Assuntos
Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/química , Interações Hospedeiro-Patógeno/genética , Conformação Proteica , Fatores de Virulência/química , Animais , Apoptose/genética , Arginina/química , Arginina/genética , Coenzima A Ligases/química , Coenzima A Ligases/genética , Cristalografia por Raios X , Domínio de Morte/genética , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/genética , Guanidina/química , Humanos , Manganês/química , Camundongos , Mutagênese , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Fatores de Virulência/genética
14.
Nucleic Acids Res ; 47(9): 4859-4871, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30892606

RESUMO

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Fator de Processamento U2AF/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Processamento Alternativo/genética , Motivos de Aminoácidos/genética , Arginina/genética , Regulação Viral da Expressão Gênica/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Ligação Proteica/genética , Fatores de Processamento de RNA/genética , Spliceossomos/genética , Replicação Viral/genética
15.
Sci Total Environ ; 667: 563-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30833255

RESUMO

Microcystin-leucine arginine (MC-LR) which is produced by cyanobacteria is a potent toxin for the reproductive system. Our previous work has demonstrated that both acute and chronic reproductive toxicity engendered by MC-LR can result in the decline of sperm quality and damage of testicular structures in male mice. The present study was designed to investigate the impact of chronic low-dose exposure to MC-LR on the regulation of RNA networks including mRNA, microRNA (miRNA), piwi-associated RNA (piRNA), covalently closed circular RNA (circRNA) and long non-coding RNA (lncRNA) in testicular tissues. By high-throughput sequencing analysis, 1091 mRNAs, 21 miRNAs, 644 piRNAs, 278 circRNAs and 324 lncRNAs were identified to be significantly altered in testicular tissues treated with MC-LR. We performed gene ontology (GO) analysis to ascertain the biological functions of differentially expressed genes. Among the altered 21 miRNAs and 644 piRNAs, the miRNA chr13_8977, which is a newly discovered species, and the piRNA mmu_piR_027558 were dramatically down-regulated after exposure to MC-LR. Consistently, both mRNA levels and protein expression levels of their predicted targets were increased significantly when chr13_8977 and mmu_piR_027558 were each down-regulated. Testicular structures, germ cell apoptosis and sperm quality were also affected by the altered expression of chr13_8977 and mmu_piR_027558 severally. We further investigated the differential expression of circRNAs and lncRNAs and their biological functions in testicular tissues following treatment with chronic low-dose exposure to MC-LR. We also constructed a competing endogenous RNA (ceRNA) network to predict the functions of the altered expressed RNAs using MiRanda. Our study suggested a crucial role for the potential network regulation of miRNAs, piRNAs, circRNAs, lncRNAs and mRNAs impacting the cytotoxicity of MC-LR in testicular tissues, which provides new perspectives in the development of diagnosis and treatment strategies for MC-LR-induced male reproductive toxicity.


Assuntos
Arginina/genética , Leucina/genética , Microcistinas/toxicidade , Reprodução/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Masculino , Camundongos , MicroRNAs , RNA , RNA Longo não Codificante , RNA Mensageiro , RNA Interferente Pequeno , Transcriptoma
16.
Infect Genet Evol ; 71: 51-53, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30898642

RESUMO

The arginine catabolic mobile element (ACME) was first described in methicillin-resistant Staphylococcus aureus and is considered to enhance transmission, persistence and survival. Subsequently ACMEs were shown to be more prevalent in the coagulase-negative Staphylococcus epidermidis. Previously, ACME types were distinguished by characteristic combinations of the arc and opp3 operons [I (arc+, opp3+), II (arc+, opp3-) and III (arc-, opp3+)] encoding an arginine deaminase pathway and oligopeptide permease transporter, respectively. Recently two novel ACME types harboring the potassium transporter-encoding operon kdp were described in oral S. epidermidis isolates [IV (arc+, opp3-, kdp+), and V (arc+, opp3+, kdp+)]. This study investigated two independent oral S. epidermidis isolates that yielded amplimers with kdp-directed primers only when subjected to ACME typing PCRs. Hybrid assemblies based on Illumina MiSeq short-read and Oxford Nanopore MinION long-read whole genome sequences revealed that both isolates harbored a sixth, novel ACME type (VI) integrated into orfX. Both ACME VIs lacked the arc and opp3 operons, harbored the kdp operon adjacent to other commonly ACME-associated genes including speG, hsd, sdr, and rep, but the structural organization of the adjacent regions were distinct. These ACMEs were flanked by different direct repeat sequences and the ACME VI-positive isolates belonged to unrelated genetic clusters. Overall these findings are indicative of independent evolution. The identification of ACME type VI further illustrates the diversity of ACME elements in S. epidermidis. The presence of ACMEs harboring kdp may confer a selective advantage on oral S. epidermidis in a potassium-rich environment such as found in dental plaque.


Assuntos
Arginina/genética , Elementos de DNA Transponíveis/genética , Staphylococcus epidermidis/genética , Adenosina Trifosfatases/genética , Arginina/metabolismo , Proteínas de Transporte de Cátions/genética , Variação Genética , Ilhas Genômicas , Humanos , Óperon/genética , Infecções Estafilocócicas/microbiologia , Sequenciamento Completo do Genoma
18.
BMC Mol Biol ; 20(1): 5, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755162

RESUMO

BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.


Assuntos
Sequência Conservada , Evolução Molecular , Proteínas Ativadoras de GTPase/química , Tubarões/metabolismo , Proteínas rab de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/genética , Cristalografia por Raios X , Glicina/química , Glicina/genética , Humanos , Lisina/química , Lisina/genética , Camundongos , Mutação , Domínios Proteicos , Alinhamento de Sequência , Tubarões/genética , Especificidade por Substrato , Suínos , Proteínas rab de Ligação ao GTP/genética
19.
Int J Cardiol ; 282: 76-80, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30772011

RESUMO

BACKGROUND: The inhibitory subunit of cardiac troponin (cTnI) is a gold standard cardiac biomarker and also an essential protein in cardiomyocyte excitation-contraction coupling. The interactions of cTnI with other proteins are fine-tuned by post-translational modification of cTnI. Mutations in cTnI can lead to hypertrophic cardiomyopathy. METHODS AND RESULTS: Here we report, for the first time, that cTnI is modified by arginine methylation in human myocardium. Using Western blot, we observed reduced levels of cTnI arginine methylation in human hypertrophic cardiomyopathy compared to dilated cardiomyopathy biopsies. Similarly, using a rat model of cardiac hypertrophy we observed reduced levels of cTnI arginine methylation compared to sham controls. Using mass spectrometry, we identified cTnI methylation sites at R74/R79 and R146/R148 in human cardiac samples. R146 and R148 lie at the boundary between the critical cTnI inhibitory and switch peptides; PRMT1 methylated an extended inhibitory peptide at R146 and R148 in vitro. Mutations at R145 that have been associated with hypertrophic cardiomyopathy hampered R146/R148 methylation by PRMT1 in vitro. H9c2 cardiac-like cells transfected with plasmids encoding for a methylation-deficient R146A/R148A cTnI protein developed cell hypertrophy, with a 32% increase in cell size after 72 h, compared to control cells. DISCUSSION: Our results provide evidence for a novel and significant cTnI post-translational modification. Our work opens the door to translational investigations of cTnI arginine methylation as a biomarker of disease, which can include e.g. cardiomyopathies, myocardial infarction and heart failure, and offers a novel way to investigate the effect of cTnI mutations in the inhibitory/switch peptides.


Assuntos
Arginina/genética , Arginina/metabolismo , Miocárdio/metabolismo , Troponina I/genética , Troponina I/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Masculino , Metilação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley
20.
Glia ; 67(4): 759-774, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30623988

RESUMO

Astrocytes respond to energetic demands by upregulating glycolysis, lactate production, and respiration. This study addresses the role of respiration and calcium regulation of respiration as part of the astrocyte response to the workloads caused by extracellular ATP and glutamate. Extracellular ATP (100 µM to 1 mM) causes a Ca2+ -dependent workload and fall of the cytosolic ATP/ADP ratio which acutely increases astrocytes respiration. Part of this increase is related to a Ca2+ -dependent upregulation of cytosolic pyruvate production. Conversely, glutamate (200 µM) causes a Na+ , but not Ca2+ , dependent workload even though glutamate-induced Ca2+ signals readily reach mitochondria. The glutamate workload triggers a rapid fall in the cytosolic ATP/ADP ratio and stimulation of respiration. These effects are mimicked by D-aspartate a nonmetabolized agonist of the glutamate transporter, but not by a metabotropic glutamate receptor agonist, indicating a major role of Na+ -dependent workload in stimulated respiration. Glutamate-induced increase in respiration is linked to a rapid increase in glycolytic pyruvate production, suggesting that both glutamate and extracellular ATP cause an increase in astrocyte respiration fueled by workload-induced increase in pyruvate production. However, glutamate-induced pyruvate production is partly resistant to glycolysis blockers (iodoacetate), indicating that oxidative consumption of glutamate also contributes to stimulated respiration. As stimulation of respiration by ATP and glutamate are similar and pyruvate production smaller in the first case, the results suggest that the response to extracellular ATP is a Ca2+ -dependent upregulation of respiration added to glycolysis upregulation. The global contribution of astrocyte respiratory responses to brain oxygen consumption is an open question.


Assuntos
Trifosfato de Adenosina/farmacologia , Astrócitos/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Mitocôndrias/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Arginina/análogos & derivados , Arginina/genética , Arginina/metabolismo , Astrócitos/ultraestrutura , Cálcio/metabolismo , Células Cultivadas , Cumarínicos/metabolismo , Líquido Extracelular/efeitos dos fármacos , Feminino , Glucose/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Sódio/metabolismo
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