RESUMO
In yeast, ras-like small G proteins, Gtr1p and Gtr2p, form heterodimers that affect cell division, detect amino acids, and regulate the activity of TORC1, a protein complex that integrates various signals, including those related to nutrient availability, growth factors, and stress signals. To explore novel roles of Gtr2p, yeast two-hybrid screening was performed using gtr2S23Np, an active form of Gtr2p, which identified Rps31p and Rpl12p as Gtr2p-interacting proteins. In the present study, we found that Gtr2p, but not Gtr1p, interacts with Rps31p, a 40S ribosomal subunit, and a component of the ubiquitin fusion protein Ubi3p, which is essential for the initiation and elongation of translation. In yeast cells expressing gtr2Q66Lp, an inactive form of Gtr2p, the interaction between Rps31p and gtr2Q66Lp, as well as the level of exogenous expression of Rps31p, was reduced. However, the level of exogenous expression of Rpl12p was unaffected. Introducing a mutation in ubiquitin target lysine residues to arginine (rps31-K5R) restored the level of exogenously expressed Rps31p and rescued the rapamycin and caffeine sensitivity of gtr2Q66L cells. Sucrose density gradient centrifugation of yeast cell lysate expressing Rps31p and gtr2Q66Lp revealed that exogenously expressed Rps31p was poorly incorporated, whereas rps31-K5Rp was efficiently incorporated, into ribosomes. These results suggest that Gtr2p influences incorporation of Rps31p into ribosomes and contributes to drug resistance through its interaction with Rps31p.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismoRESUMO
Yeast Gtr1p is a GTPase that forms a heterodimer with Gtr2p, another GTPase; it is involved in regulating TORC1 activity in nutrient signaling, including amino acid availability and growth control. Gtr1p is a positive regulator of TORC1, a kinase that regulates various cellular functions (e.g., protein synthesis and autophagy) under specific nutrient and environmental conditions, including oxidative stress. In this study, we examined the roles of Gtr1p in oxidative stress responses. We found that yeast cells expressing guanosine diphosphatase (GDP)-bound Gtr1p (Gtr1-S20Lp) were resistant to hydrogen peroxide (H2O2), whereas guanosine triphosphate (GTP)-bound Gtr1p (Gtr1-Q65Lp) was sensitive to H2O2 compared with the wild type. Consistent with these findings, yeast cells lacking Iml1p, a component of the GTPase-activating protein complex for Gtr1p, exhibited the H2O2-sensitive phenotype. In gtr1S20L cells, autophagy was highly induced under oxidative stress. gtr1Q65L cells showed decreased expression of the SNQ2 gene, which encodes a multidrug transporter involved in resistance to oxidative stress, and the overexpression of SNQ2 rescued the oxidative stress sensitivity of gtr1Q65L cells. These results suggest that Gtr1p is involved in oxidative stress responses through mechanisms that include autophagy and SNQ2 expression.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/metabolismo , Estresse Oxidativo/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Autofagia , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Proteínas Monoméricas de Ligação ao GTP/genética , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
WDR35/IFT121 is an intraflagellar transport protein in primary cilia, which is associated with RagA, an mTORC1-activating protein. To elucidate the functions of the interaction between WDR35 and RagA in primary cilia, as well as mTOR signaling, we identified WDR35-interacting proteins using mass spectrometry. We found that WDR35 associates with CCT complex proteins including TCP1/CCT1, which act as molecular chaperones for α-tubulin folding. Immunostaining showed that acetylated α-tubulin was concentrated in the vicinity of primary cilia in 293T cells. In contrast, acetylated tubulin was dispersed in WDR35 partial knockout cells established from 293T cells. Similarly, scattered subcellular localization of acetylated tubulin was observed in RagA knockout cells. RagA was present in the primary cilia of NIH3T3 cells, and the GDP form of RagA exhibited strong binding to WDR35 and negative regulation of primary cilium formation. These results suggest that WDR35 is involved in the subcellular localization of acetylated tubulin in primary cilia via its interactions with TCP1 and/or RagA family proteins.
Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Células Cultivadas , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo , Tubulina (Proteína)/químicaRESUMO
Small Ras-like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras-like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over-expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA-, RagB- and RagC-dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Proteínas do Citoesqueleto , Células HEK293 , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/genética , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions. While AUF1 binds to oligoribonucleotides carrying a single 8-oxoG, PCBP1 does not bind to such oligoribonucleotides but instead binds firmly to oligoribonucleotides in which two 8-oxoG residues are located nearby. PCBP1-deficient cells, constructed from the human HeLa S3 line using the CRISPR-Cas9 system, exhibited higher survival rates than HeLa S3 cells when small doses of hydrogen peroxide were applied. The levels of caspase-3 activation and PARP-1 cleavage in the PCBP1-deficient cells were significantly lower than those in wild-type cells. The structure-function relationship of PCBP1 was established with the use of PCBP1 mutant proteins in which the conserved KH domains were defective. Human cells appear to possess two distinct mechanisms, one controlled by AUF1 and the other by PCBP1, with the former functioning when messenger RNA is moderately oxidized and the latter operating when the RNA is more severely damaged.
Assuntos
Apoptose/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , RNA Mensageiro/metabolismo , Substituição de Aminoácidos , Sistemas CRISPR-Cas , Caspase 3/fisiologia , Sequência Conservada , Proteínas de Ligação a DNA , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/química , Humanos , Peróxido de Hidrogênio/farmacologia , Oxirredução , Domínios Proteicos , RNA Mensageiro/química , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/fisiologia , Estresse Oxidativo , RNA Mensageiro/metabolismo , Linhagem Celular , Ribonucleoproteína Nuclear Heterogênea D0 , HumanosRESUMO
The yeast Ras-like GTPases Gtr1p and Gtr2p form a heterodimer, are implicated in the regulation of TOR complex 1 (TORC1) and play pivotal roles in cell growth. Gtr1p and Gtr2p bind Ego1p and Ego3p, which are tethered to the endosomal and vacuolar membranes where TORC1 functions are regulated through a relay of amino acid signaling interactions. The mechanisms by which Gtr1p and Gtr2p activate TORC1 remain obscure. We probed the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 subunits. Mutations in the region (179-220 a.a.) following the nucleotide-binding region of Gtr1p and Gtr2p abrogated their mutual interaction and resulted in a loss in function, suggesting that complex formation between Gtr1p and Gtr2p was indispensable for TORC1 function. A modified yeast two-hybrid assay showed that Gtr1p-Gtr2p complex formation is important for its interaction with the Ego1p-Ego3p complex. GTP-bound Gtr1p interacted with the region containing the HEAT repeats of Kog1p and the C-terminal region of Tco89p. The GTP-bound Gtr2p suppressed a Kog1p mutation. Our findings indicate that the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 components Kog1p and Tco89p play a role in TORC1 function.
Assuntos
Aminoácidos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The yeast ubiquitin-like and ubiquitin-associated protein Dsk2 is one of the ubiquitin receptors that function in the ubiquitin-proteasome pathway. We screened the Dsk2-interacting proteins in Saccharomyces cerevisiae by a two-hybrid assay and identified a novel Dsk2-interacting protein, Irc22, the gene locus of which has previously been described as YEL001C, but the function of which is unknown. IRC22/YEL001C encodes 225 amino acid residues with a calculated molecular weight of 25 kDa. The Irc22 protein was detected in yeast cells. IRC22 was a nonessential gene for yeast growth, and its homologs were found among ascomycetous yeasts. Irc22 interacted with Dsk2 in yeast cells, but not with Rad23 and Ddi1. Ubiquitin-dependent degradation was impaired mildly by over-expression or disruption of IRC22. Compared with the wild-type strain, dsk2D exhibited salt sensitivity while irc22D exhibited salt tolerance at high temperatures. The salt-tolerant phenotype that was observed in irc22D disappeared in the dsk2Dirc22D double disruptant, indicating that DSK2 is positively and IRC22 is negatively involved in salt stress tolerance. IRC22 disruption did not affect any responses to DNA damage and oxidative stress when comparing the irc22D and wild-type strains. Collectively, these results suggest that Dsk2 and Irc22 are involved in salt stress tolerance in yeast.
RESUMO
In aerobically growing cells, in which reactive oxygen species are produced, the guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine as well as cytosine. This mispairing causes alterations in gene expression, and cells possess mechanisms to prevent such outcomes. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced by mutations in genes related to the prevention of abnormal protein synthesis under oxidative stress. A genome-wide search for the genes responsible, followed by DNA sequence determination, revealed that specific amino acid changes in guanylate kinase and in the ß and ß' subunits of RNA polymerase cause elevated levels of phenotypic suppression, specifically under aerobic conditions. The involvement of the DnaB, DnaN, and MsbA proteins, which are involved in DNA replication and in preserving the membrane structure, was also noted. Interactions of these proteins with each other and also with other molecules may be important for preventing errors in gene expression.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Guanilato Quinases/metabolismo , Estresse Oxidativo/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , RNA Polimerases Dirigidas por DNA/genética , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Estudo de Associação Genômica Ampla , Guanina/análogos & derivados , Guanina/metabolismo , Guanilato Quinases/genética , OxirreduçãoRESUMO
Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.
Assuntos
Nucleotídeos de Desoxiguanina/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/biossíntese , Trifosfato de Adenosina/química , Adenilato Quinase/química , Citidina Trifosfato/química , Nucleotídeos de Desoxiguanina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Guanosina Monofosfato/química , Guanilato Quinases/química , Cinética , Núcleosídeo-Difosfato Quinase/química , Oxirredução , Pirofosfatases/química , RNA Bacteriano/metabolismo , Uridina Trifosfato/químicaRESUMO
Chlamydomonas reinhardtii α-type carbonic anhydrase (Cr-αCA1) is a dimeric enzyme that catalyses the interconversion of carbon dioxide and carbonic acid. The precursor form of Cr-αCA1 undergoes post-translational cleavage and N-glycosylation. Comparison of the genomic sequences of precursor Cr-αCA1 and other αCAs shows that Cr-αCA1 contains a different N-terminal sequence and two insertion sequences. A 35-residue peptide in one of the insertion sequences is deleted from the precursor during maturation. The crystal structure of the mature form of Cr-αCA1 has been determined at 1.88â Å resolution. Each subunit is cleaved into the long and short peptides, but they are linked together by a disulfide bond. The two subunits are linked by a disulfide bond. N-Glycosylations occur at three asparagine residues and the attached N-glycans protrude into solvent regions. The subunits consist of a core ß-sheet structure composed of nine ß-strands. At the centre of the ß-sheet is the catalytic site, which contains a Zn atom bound to three histidine residues. The amino-acid residues around the Zn atom are highly conserved in other monomeric and dimeric αCAs. The short peptide runs near the active site and forms a hydrogen bond to the zinc-coordinated residue in the long chain, suggesting an important role for the short peptide in Cr-αCA1 activity.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Chlamydomonas reinhardtii/enzimologia , Sequência de Aminoácidos , Asparagina/metabolismo , Anidrases Carbônicas/genética , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Dimerização , Dissulfetos/química , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Zinco/metabolismoRESUMO
Ubiquitin-like (UBL)-ubiquitin-associated (UBA) proteins, including Dsk2 and Rad23, act as delivery factors that target polyubiquitinated substrates to the proteasome. We report here that the Dsk2 UBL domain is ubiquitinated in yeast cells and that Dsk2 ubiquitination of the UBL domain is involved in Dsk2 stability, depending on the Dsk2 UBA domain. Also, Dsk2 lacking ubiquitin chains impaired ubiquitin-dependent protein degradation and decreased the interaction of Dsk2 with polyubiquitinated proteins in cells. Moreover, Dsk2 ubiquitination affected ability to restore the temperature-sensitive growth defect of dsk2Δ. These results indicate that ubiquitination in the UBL domain of Dsk2 has in vivo functions in the ubiquitin-proteasome pathway in yeast.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
Carbonic anhydrases (CAs) are ubiquitously distributed and are grouped into three structurally independent classes (alphaCA, betaCA and gammaCA). Most alphaCA enzymes are monomeric, but alphaCA1 from Chlamydomonas reinhardtii is a dimer that is uniquely stabilized by disulfide bonds. In addition, during maturation an internal peptide of 35 residues is removed and three asparagine residues are glycosylated. In order to obtain insight into the effects of these structural features on CA function, wild-type C. reinhardtii alphaCA1 has been crystallized in space group P6(5), with unit-cell parameters a=b=134.3, c=120.2 A. The crystal diffracted to 1.88 A resolution and a preliminary solution of its crystal structure has been obtained by the MAD method.
Assuntos
Anidrases Carbônicas/química , Chlamydomonas reinhardtii/enzimologia , Cristalografia por Raios XRESUMO
Actinohivin (AH) is a microbial lectin containing 114 amino acids, which inhibits human immunodeficiency virus (HIV) infection. This effect is brought about by its specific binding to Man-α(1-2)-Man unit(s) of high-mannose type glycan (HMTG) bound to HIV gp120. The recently determined crystal structure of AH suggests that three repeated segments (the residue numbers 1-38, 39-76 and 77-114 for segments 1, 2 and 3, respectively) form three sugar-binding pockets to accommodate Man-α(1-2)-Man units. The strong specific binding of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the 'cluster effect' of lectin. It remains to be seen which residues of the sugar-binding pockets are essential for acceptance of Man-α(1-2)-Man. To identify the amino acid residues critical for anti-HIV effect, we performed mutational analysis. Mutant AHs were subjected to enzyme-linked immunosorbent assay testing for gp120-binding activity and to syncytium formation assay. As a result, it was revealed that Asp15, Tyr23, Leu25, Asn28 and Tyr32 in segment 1, Tyr61 in segment 2 and Tyr99 in segment 3 are essential for anti-HIV activity. The conserved residues, Asp53, Leu63, Asn66 and Tyr70, in segment 2 and, Asp91, Leu101, Asn104 and Tyr108, in segment 3 are also necessary. Furthermore, aromatic residues at positions 23 and 32 are required for creation of potency. These data will be useful for predicting the detailed mechanism of AH-Man-α(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas de Bactérias/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV/efeitos dos fármacos , Sequência de Aminoácidos , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Manose/metabolismo , Mutação , Ligação ProteicaRESUMO
The alkaline treatment and dehydrogenation of pyridinium salts, formed from the S-alkylations of 3-(1-pyridinio)thiophene-2-thiolates with alpha,alpha-dibromo-o-, m-, or p-xylene, provided the corresponding alpha,alpha'-bis[(thieno[3,4-b]indolizin-3-yl)thio]-o-, m-, and p-xylene derivatives in low to good yields. Both (1)H-NMR and UV-Vis spectra of these products supported distinctly the predominance of the gauche-gauche conformation in relation to the two sulfide linkages as the spacer in these molecules. On the other hand, the X-ray analyses indicated the expected gauche-gauche conformation for the m- and the p-xylene derivatives, but the anti-anti one for the o-xylene derivative.
Assuntos
Compostos Heterocíclicos/química , Compostos Heterocíclicos/síntese química , Indolizinas/química , Indolizinas/síntese química , Nitrogênio , Compostos de Sulfidrila/química , Compostos de Sulfidrila/síntese química , Xilenos/química , Xilenos/síntese química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Nitrogênio/químicaRESUMO
Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man alpha(1-2)Man, Man alpha(1-2)Man alpha(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K(d) = 3.4 x 10(-8) M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the "cluster effect" of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.
Assuntos
Proteínas de Bactérias/farmacologia , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Lectinas/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacocinética , Sítios de Ligação , Proteínas de Transporte/farmacocinética , Proteínas de Transporte/farmacologia , Cristalografia por Raios X , Proteína gp120 do Envelope de HIV/química , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/farmacocinética , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Cinética , Lectinas/química , Lectinas/farmacocinética , Manose/química , Manosídeos/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA(Thr) from different tRNAs in order to generate Thr-tRNA(Thr). In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA(Thr), one for catalysis and the other for trans-editing of misacylated Ser-tRNA(Thr). In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 A resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.
Assuntos
Aeropyrum/metabolismo , RNA Arqueal/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Treonina-tRNA Ligase/química , Aminoacilação de RNA de Transferência , Aeropyrum/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Treonina/metabolismo , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismoRESUMO
RNA 3'-terminal phosphate cyclase (Rtc) is an enzyme involved in RNA splicing that converts the 3'-terminal hydroxyl group of truncated RNA to 2',3'-cyclic phosphate, which is required just before its ligation. This reaction may occur in the following two steps: (i) Rtc + ATP --> Rtc-AMP + PP(i) and (ii) RNA-N3'p + Rtc-AMP --> RNA-N>p + Rtc + AMP. In order to reveal the reaction mechanism, Rtc of Sulfolobus tokodaii (St-Rtc) overexpressed in Escherichia coli was purified and crystallized in the following states: St-Rtc, St-Rtc+Mn, St-Rtc+ATP, St-Rtc+AMP and St-Rtc-AMP. The crystals diffracted to 2.25-3.00 A resolution and preliminary solutions of their structures have been obtained by molecular replacement using the structure of a selenomethionine-labelled St-Rtc crystal which was solved in advance using the MAD method as a model. These crystals grew in two different space groups (P3(1) and P4(2)), with the former space group displaying two distinct packing modes.
Assuntos
Proteínas Arqueais/química , Ligases/química , Sulfolobus/enzimologia , Sequência de Aminoácidos , Apoenzimas/química , Proteínas Arqueais/isolamento & purificação , Soluções Tampão , Crenarchaeota/enzimologia , Cristalização , Dimerização , Escherichia coli/genética , Vetores Genéticos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Ligases/isolamento & purificação , Ligases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rotação , Estatística como Assunto , Transformação Bacteriana , Difração de Raios XRESUMO
D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.
Assuntos
Alcaligenes faecalis/enzimologia , Hidroxibutirato Desidrogenase/química , Ácido 3-Hidroxibutírico/química , Acetoacetatos/química , Biocatálise , Coenzimas , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , NAD/química , Eletricidade Estática , Especificidade por SubstratoRESUMO
The yeast Ras-like small GTPases Gtr1p and Gtr2p form a heterodimer and interact genetically with Prp20p, a guanine nucleotide exchange factor for the GTPase Gsp1p. Gtr1p and Gtr2p may be involved in nucleocytoplasmic transport and in the nutrient-responsive TOR signaling pathway, but the role of the Gtr1p-Gtr2p heterodimer is not well understood. Characterization of the Gtr1p-Gtr2p complex is indispensable for understanding the functions of both Gtr1p and Gtr2p. We analyzed the association mode between Gtr1p and Gtr2p. The N-terminus nucleotide binding region of Gtr1p associated with Gtr2p, but not with Ego1p, a protein known to interact with Gtr1p. Gtr1p and Gtr2p are necessary for cells to acquire resistance to caffeine, rapamycin, and hydrogen peroxide. Caffeine treatment released Gtr1p from the high molecular weight Gtr1p-Gtr2p complex. Gtr2p mutants S23N and T44N, but not Q66L, rescued the gtr2 disruptant. Our findings indicate that the formation of heterodimers by Gtr1p differs between Gtr2p and Ego1p.