Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.441
Filtrar
2.
Mol Pharmacol ; 98(6): 710-718, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33008919

RESUMO

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.


Assuntos
Arginina/genética , Glucuronosiltransferase/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Sítios de Ligação/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Glucosiltransferases/genética , Glucosiltransferases/ultraestrutura , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Glicosídeos/metabolismo , Células HEK293 , Humanos , Himecromona/metabolismo , Medicago truncatula , Simulação de Dinâmica Molecular , Morfina/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , Zidovudina/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(40): 25104-25115, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958650

RESUMO

Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteoma/genética , Receptores Notch/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Arginina/genética , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais/genética
4.
Nucleic Acids Res ; 48(18): 10280-10296, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32955564

RESUMO

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.


Assuntos
Arginina/análogos & derivados , Fator de Iniciação 2 em Eucariotos/genética , RNA Mensageiro/genética , Substituição de Aminoácidos/genética , Arginina/genética , Códon de Iniciação/genética , Humanos , Complexos Multiproteicos/genética , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética
5.
Nat Med ; 26(10): 1602-1608, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32747827

RESUMO

Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa1-4. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda7. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.


Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Mutação de Sentido Incorreto , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Substituição de Aminoácidos/genética , Animais , Arginina/genética , Evolução Clonal/genética , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/parasitologia , Genótipo , Histidina/genética , Humanos , Técnicas In Vitro , Repetição Kelch/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Testes de Sensibilidade Parasitária , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético , Proteínas de Protozoários/química , Ruanda/epidemiologia
6.
Mol Pharmacol ; 98(2): 143-155, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32616523

RESUMO

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.


Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Arginina/genética , Sítios de Ligação/efeitos dos fármacos , Células CHO , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mutação , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Ratos , Xenopus
7.
Proc Natl Acad Sci U S A ; 117(27): 15731-15739, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561643

RESUMO

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.


Assuntos
Arginina/química , Nucleoproteínas/genética , Ornitina/química , Peptídeos/genética , Sequência de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Arginina/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/química , Ornitina/genética , Peptídeos/química , Proteínas/química , Proteínas/genética , RNA/química , RNA/genética
8.
Sci Rep ; 10(1): 7817, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385379

RESUMO

The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA - C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.


Assuntos
Arginina/genética , DNA Girase/genética , Conformação Proteica em Folha beta/genética , Salmonella typhi/genética , Sequência de Aminoácidos/genética , DNA Girase/ultraestrutura , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Salmonella typhi/enzimologia , Salmonella typhi/patogenicidade
9.
Proc Natl Acad Sci U S A ; 117(18): 9876-9883, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303654

RESUMO

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex.


Assuntos
Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/ultraestrutura , Proteínas de Transporte/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Esclerose Amiotrófica Lateral/genética , Arginina/genética , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteína C9orf72/genética , Proteínas de Transporte/genética , Microscopia Crioeletrônica , Filaminas/genética , Filaminas/ultraestrutura , Demência Frontotemporal/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/ultraestrutura , Predisposição Genética para Doença , Humanos , Complexos Multiproteicos/genética , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/genética
10.
Biochim Biophys Acta Gen Subj ; 1864(7): 129597, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32156582

RESUMO

The arginine repressor (ArgR) regulates the expression of genes involved in arginine biosynthesis. Upon attaining a threshold concentration of arginine in the cytoplasm, the trimeric C-terminal domain of ArgR binds three arginines in a shallow surface cleft and subsequently hexamerizes forming a dimer of trimers containing six Arg co-repressor molecules which are buried at the subunit interfaces. The N-terminal domains of this complex bind to the DNA promoter thereby interrupting the transcription of the genes related to Arg biosynthesis. The crystal structures of the wild type and mutant Pro115Gln ArgR from Corynebacterium pseudotuberculosis determined at 1.7 Å demonstrate that a single amino acid substitution switches co-repressor specificity from Tyr to Arg. Molecular dynamics simulations indicate that the first step, i.e., the binding of the co-repressor, occurs in the trimeric state and that Pro115Gln ArgR preferentially binds Arg. It was also shown that, in Pro115 ArgR hexamers, the concomitant binding of sodium ions shifts selectivity to Tyr. Structural data combined with phylogenetic analyses of ArgR from C. pseudotuberculosis suggest that substitutions in the binding pocket at position 115 may alter its specificity for amino acids and that the length of the protein interdomain linker can provide further functional flexibility. These results support the existence of alternative ArgR regulatory mechanisms in this pathogenic bacterium.


Assuntos
Proteínas de Bactérias/genética , Corynebacterium pseudotuberculosis/genética , Filogenia , Proteínas Repressoras/genética , Transcrição Genética , Sequência de Aminoácidos/genética , Arginina/biossíntese , Arginina/genética , Sítios de Ligação , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética
11.
Eur J Med Chem ; 192: 112199, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32171162

RESUMO

A series of PROTAC (proteolysis targeting chimera) based selective EGFRL858R/T790M (leucine 858 to arginine 858 mutation and threonine 790 to methionine 790) mutant degraders were designed and synthesized. One of the most potent compounds, 14o, effectively and selectively degraded EGFRL858R/T790M with an DC50 value of 5.9 nM, while did not show obvious effect on the wild-type protein. Further mechanism investigation revealed that the degradation was mediated by ubiquitin proteasome pathway. Compound 14o could be utilized as an initial lead molecule for development of new EGFRL858R/T790M degrader based therapy.


Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Arginina/genética , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Leucina/genética , Metionina/genética , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Treonina/genética , Células Tumorais Cultivadas
12.
Asian Pac J Cancer Prev ; 21(3): 663-665, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32212791

RESUMO

BACKGROUND AND OBJECTIVE: X-ray repair cross-complementing group1 (XRCC1) is a key protein in base excision repair and closely associated with the coordination of the base excision repair pathway. Many studies have focused on XRCC1 SNPs and have shown an associated between these SNPs and the risk of several types of cancers, including head and neck cancer. There are many single nucleotide polymorphisms XRCC1 gene (SNPs) and the most common SNP that result in amino acid substitutions is exon 10 (Arg399Gln). This study aimed to investigate the association between Arg399Gln SNP and the risk of squamous cell carcinoma of the head and neck. MATERIAL AND METHOD: Ninety nine patients with squamous cell carcinomas of the head and neck and 89 healthy adult controls were enrolled in this study. The Arg399Gln in XRCC1 allele was genotyped using polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: In the single-locus analyses, Arg399Gln SNP showed a significant association with head and neck cancer risk (p value = 0.016 and odd ratio of 1.8). On the genotype level, we applied three analysis models, namely co-dominant, dominant, and recessive genotypes. Arg/Arg homozygous major genotype was significantly (p value <0.05) associated with head and neck squamous cell carcinoma incidence with odd ratio of 2.23 and 2.24 for the co-dominant and recessive models, respectively. CONCLUSION: The findings indicated that Arg399Gln allele was associated with squamous cell carcinoma of the head and neck among Jordanian patients. This allele might be used as a genetic biomarker of squamous cell carcinoma of the head and neck.


Assuntos
Neoplasias de Cabeça e Pescoço/genética , Polimorfismo de Nucleotídeo Único , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Adulto , Idoso , Arginina/genética , Feminino , Glutamina/genética , Humanos , Masculino , Pessoa de Meia-Idade
13.
Biochem Pharmacol ; 174: 113850, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32044355

RESUMO

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.


Assuntos
Arginina/metabolismo , Asparagina/metabolismo , Simulação por Computador , Família 4 do Citocromo P450/metabolismo , Arginina/química , Arginina/genética , Asparagina/química , Asparagina/genética , Sítios de Ligação/fisiologia , Família 4 do Citocromo P450/química , Família 4 do Citocromo P450/genética , Humanos , Mutação de Sentido Incorreto/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato/fisiologia
14.
Mol Cell ; 77(6): 1237-1250.e4, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32048997

RESUMO

Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing.


Assuntos
Arginina/metabolismo , Núcleo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Lisina/metabolismo , Processamento de RNA/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Arginina/genética , Núcleo Celular/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Lisina/genética , Mutação , Fosforilação , Domínios Proteicos , RNA Mensageiro/genética , Fatores de Processamento de Serina-Arginina/genética
15.
Mol Pharmacol ; 97(4): 267-277, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32005759

RESUMO

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 Half-maximal effective potential (V0.5) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.


Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Arginina/genética , Sítios de Ligação/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Iloprosta/farmacologia , Ligantes , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Receptores de Epoprostenol/metabolismo , Receptores de Prostaglandina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
16.
Sci Rep ; 10(1): 2000, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029872

RESUMO

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.


Assuntos
Distrofias Hereditárias da Córnea/genética , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fator de Crescimento Transformador beta/genética , Substituição de Aminoácidos , Animais , Arginina/genética , Sistemas CRISPR-Cas , Distrofias Hereditárias da Córnea/patologia , Substância Própria/patologia , Substância Própria/ultraestrutura , Cisteína/genética , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação , Reparo de DNA por Recombinação
17.
J Biol Chem ; 295(15): 4822-4835, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094223

RESUMO

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.


Assuntos
Arginina/metabolismo , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Arginina/química , Arginina/genética , Movimento Celular , Células HEK293 , Humanos , Lisina/química , Lisina/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética
18.
Amino Acids ; 52(1): 103-110, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31832896

RESUMO

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.


Assuntos
Anticorpos/farmacologia , Arginina/genética , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fator de Necrose Tumoral alfa/genética , Animais , Anticorpos/imunologia , Glicemia/genética , Citrulinação/efeitos dos fármacos , Citrulinação/genética , Metilação de DNA/genética , Metilação de DNA/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T alfa-beta/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Baço/efeitos dos fármacos , Baço/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
19.
Oncogene ; 39(3): 703-717, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541192

RESUMO

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/ß), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/genética , Proteínas Recombinantes/genética , Substituição de Aminoácidos , Arginina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Membrana/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Transfecção , Tirosina/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
FEBS Lett ; 594(2): 301-316, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541584

RESUMO

Protein arginine methyltransferase 1 (PRMT1) stimulates erythroid differentiation, but the signaling events upstream are yet to be identified. Ca2+ plays crucial roles during erythroid differentiation. Here, we show that Ca2+ enhances methylation during induced erythroid differentiation and that Ca2+ directly upregulates the catalytic activity of recombinant PRMT1 by increasing Vmax toward the substrate heterogeneous nuclear ribonucleoprotein A2. We demonstrate that PRMT1 is essential and responsible for the effect of Ca2+ on differentiation. Depletion of Ca2+ suppresses PRMT1-mediated activation of p38α and p38α-stimulated differentiation. Furthermore, Ca2+ stimulates methylation of p38α by PRMT1. This study uncovers a novel regulatory mechanism for PRMT1 by Ca2+ and identifies the PRMT1/p38α axis as an intracellular mediator of Ca2+ signaling during erythroid differentiation.


Assuntos
Diferenciação Celular/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Ribonucleoproteínas/genética , Arginina/genética , Cálcio/metabolismo , Metilação de DNA/genética , Células Eritroides/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/genética , Transdução de Sinais/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA