RESUMO
Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.
Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico/fisiologia , Hidrólise , Especificidade por Substrato/fisiologia , Xilanos/metabolismo , Xilose/metabolismoRESUMO
SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.
Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Dimerização , HumanosRESUMO
The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.
Assuntos
Glucana 1,3-beta-Glucosidase/química , Glicosídeo Hidrolases/química , beta-Glucanas/química , Sequência de Aminoácidos/genética , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Glucana 1,3-beta-Glucosidase/metabolismo , Glucanos/química , Glicosídeos/química , Modelos Moleculares , Especificidade por Substrato/fisiologiaRESUMO
Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.
Assuntos
Domínio Catalítico/fisiologia , Leucócitos/patologia , Metaloproteases/farmacologia , Venenos de Serpentes/enzimologia , Músculos Abdominais/irrigação sanguínea , Animais , Bothrops , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Metaloproteases/química , Camundongos , MicrocirculaçãoRESUMO
Glutathione transferases (GSTs) perform several catalytic and non-catalytic roles in the defense against toxicities of electrophile compounds and oxidative stress, and therefore are involved in stress-response and cell detoxification. Previously, we have provided evidence indicating that EgGST2 and EgGST3, two phylogenetically distant Echinococcus granulosus GSTs, can naturally form a heterodimeric structure (EgGST2-3). In the present work, the recombinant heterodimer GST (rEgGST2-3) is characterized. Hence, rEgGST2-3 was able to conjugate GSH to three substrates: 1-chloro-2,4-dinitrobenzene (CDNB, general substrate for GSTs), 1,2-dichloro-4-nitrobenzene (specific substrate for mammalian Mu class) and trans,trans-deca-2,4-dienal (reactive carbonyl). The canonical activity was considerably reduced by all the conventional inhibitors (cybacron blue, triphenylthin chloride and bromosulfophthalein) and by other inhibitors (ellagic acid, alizarin and chenodeoxycholic acid). Besides this, rEgGST2-3 activity was inhibited by a number of anthelmintic drugs, where the halogenated phenolic drugs (mainly bithionol and hexachlorophene) acted as stronger inhibitors, suggesting they may bind to the EgGST2-3. Moreover, rEgGST2-3 exhibited glutathione-peroxidase activity, and its specific constant (kcat/KM) was calculated. Finally, rEgGST2-3 displayed the ability to bind non-substrate molecules, particularly anthelmintic drugs, suggesting that ligandin activity may have potential to act as a passive protection parasite mechanism. Overall, the rEgGST2-3 behavior was shown to be both complementary and redundant to that reported for rEgGST1, another characterized GST from E. granulosus. It would be appropriate that different enzymes in the same organism do not have exactly the same functional properties to develop a better adaptation to life in the host.
Assuntos
Echinococcus granulosus/enzimologia , Glutationa Transferase/fisiologia , Animais , Domínio Catalítico/fisiologia , Dinitroclorobenzeno , Echinococcus granulosus/genética , Nitrobenzenos , FilogeniaRESUMO
Epoxide hydrolases (EHs) are enzymes that have high biotechnological interest for the fine and transformation industry. Several of these enzymes have enantioselectivity, which allows their application in the separation of enantiomeric mixtures of epoxide substrates. Although two different families of EHs have been described, those that have the α/ß-hidrolase fold are the most explored for biotechnological purpose. These enzymes are functionally very well studied, but only few members have three-dimensional structures characterised. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) has been discovered and functionally studied. This enzyme does not have high homology to any other EH structure and have an enatiopreference for (S)-(-) isomers. Herein we described the crystallographic structure of TrEH at 1.7Å resolution, which reveals features of its tertiary structure and active site. TrEH has a similar fold to the other soluble epoxide hydrolases and has the two characteristic hydrolase and cap domains. The enzyme is predominantly monomeric in solution and has also been crystallised as a monomer in the asymmetric unit. Although the catalytic residues are conserved, several other residues of the catalytic groove are not, and might be involved in the specificity for substrates and in the enantioselectivy of this enzyme. In addition, the determination of the crystallographic structure of TrEH might contribute to the rational site direct mutagenesis to generate an even more stable enzyme with higher efficiency to be used in biotechnological purposes.
Assuntos
Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Trichoderma/metabolismo , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodosRESUMO
Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.
Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ligação Proteica/fisiologia , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/fisiologiaRESUMO
TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/ß region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase's active site, providing a structural framework for the function of TIPRL in PP2A inhibition.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dobramento de Proteína , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosforilação/fisiologia , Ligação Proteica/genética , Estrutura Secundária de ProteínaRESUMO
PKA (cAMP-dependent protein kinase) activity, as well as that of other AGC members, is regulated by multiple phosphorylations of its catalytic subunits. In Saccharomyces cerevisiae, the PKA regulatory subunit is encoded by the gene BCY1, and the catalytic subunits are encoded by three genes: TPK1, TPK2 and TPK3. Previously, we have reported that, following cAMP/PKA pathway activation, Tpk1 increases its phosphorylation status. Now, in vivo genetic and in vitro experiments indicate an autophosphorylation mechanism for Tpk1. Using array peptides derived from Tpk1, we identified Ser179 as a target residue. Tpk1 is phosphorylated on Ser179 in vivo during glucose stimulus. Reduction of the activation loop Thr241 phosphorylation increases Ser179 autophosphorylation. To evaluate the role of phosphorylation on Ser179, we made strains expressing tpk1S179A or tpk1S179D as the sole PKA kinase source. Our results suggest that Ser179 phosphorylation increases the reactivity towards the substrate without affecting the formation of the holoenzyme. Phenotypic readout analysis showed that Ser179 phosphorylation increases in vivo PKA activity, reducing cell survival, stress and lifespan. Ser179 phosphorylation increases Tpk1 cytoplasmic accumulation in glucose-grown cells. These results describe for the first time that an autophosphorylation mechanism on Tpk1 controls PKA activity in response to glucose availability.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínio Catalítico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fermentação , Glucose/farmacologia , Fosforilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Serina/metabolismoRESUMO
PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Rubredoxinas/metabolismo , Alcenos/química , Alcenos/metabolismo , Domínio Catalítico/fisiologia , Dicroísmo Circular , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Mutagênese Sítio-Dirigida , Nitrocompostos/química , Nitrocompostos/metabolismo , Rubredoxinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.
Assuntos
Acetiltransferases/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/metabolismo , Leveduras/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/fisiologia , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Aciltransferases/fisiologia , Sequência de Aminoácidos , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Lipoilação/genética , Proteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , Leveduras/genéticaRESUMO
In the mitochondrial F(O)F(1) ATP-synthase/ATPase complex, subunits α and ß are part of the extrinsic portion that catalyses ATP synthesis. Since there are no reports about genes and proteins from these subunits in crustaceans, we analyzed the cDNA sequences of both subunits in the whiteleg shrimp Litopenaeus vannamei and their phylogenetic relationships. We also investigated the effect of hypoxia on shrimp by measuring changes in the mRNA amounts of atpα and atpß. Our results confirmed highly conserved regions for both subunits and underlined unique features among others. The ATPß deduced protein of shrimp was less conserved in size and sequence than ATPα. The relative mRNA amounts of atpα and atpß changed in shrimp pleopods; hypoxia at 1.5 mg/L caused an increase in atpß transcripts and a subsequent decrease when shrimp were re-oxygenated. Results confirm that changes in the mRNAs of the ATP-synthase subunits are part of the mechanisms allowing shrimp to deal with the metabolic adjustment displayed to tolerate hypoxia.
Assuntos
Trifosfato de Adenosina/metabolismo , Domínio Catalítico/fisiologia , Hipóxia/enzimologia , Penaeidae/enzimologia , ATPases Translocadoras de Prótons/biossíntese , Trifosfato de Adenosina/genética , Animais , DNA Complementar/genética , DNA Complementar/metabolismo , Hipóxia/genética , Penaeidae/genética , ATPases Translocadoras de Prótons/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de Sequência de ProteínaRESUMO
The olfactory bulbs play a relevant role in the interaction between the animal and its environment. The existence of endothelin-1 and -3 in the rat olfactory bulbs suggests their role in the control of diverse functions regulated at this level. Tyrosine hydroxylase, a crucial enzyme in catecholamine biosynthesis, is tightly regulated by short- and long-term mechanisms. We have previously reported that in the olfactory bulbs endothelins participate in the short-term tyrosine hydroxylase regulation involving complex mechanisms. In the present work we studied the effect of long-term stimulation by endothelins on tyrosine hydroxylase in the rat olfactory bulbs. Our findings show that endothelin-1 and -3 modulated catecholaminergic transmission by increasing enzymatic activity. However, these peptides acted through different receptors and intracellular pathways. Endothelin-1 enhanced tyrosine hydroxylase activity through a super high affinity ET(A) receptor and cAMP/PKA and CaMK-II pathways, whereas, endothelin-3 through a super high affinity atypical receptor coupled to cAMP/PKA, PLC/PKC and CaMK-II pathways. Endothelins also increased tyrosine hydroxylase mRNA and the enzyme total level as well as the phosphorylation of Ser 19, 31 and 40 sites. Furthermore, both peptides stimulated dopamine turnover and reduced its endogenous content. These findings support that endothelins are involved in the long-term regulation of tyrosine hydroxylase, leading to an increase in the catecholaminergic activity which might be implicated in the development and/or maintenance of diverse pathologies involving the olfactory bulbs.
Assuntos
Catecolaminas/biossíntese , Endotelinas/metabolismo , Bulbo Olfatório/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/fisiologia , AMP Cíclico/metabolismo , Endotelina-1/metabolismo , Endotelina-1/farmacologia , Endotelina-3/metabolismo , Endotelina-3/farmacologia , Endotelinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Masculino , Transtornos do Olfato/metabolismo , Transtornos do Olfato/fisiopatologia , Bulbo Olfatório/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/agonistas , Receptor de Endotelina A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tempo , Fatores de Tempo , Fosfolipases Tipo C/metabolismoRESUMO
The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s(-1). We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates.
Assuntos
Domínio Catalítico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/fisiologia , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , Fosforilação/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/fisiologiaRESUMO
We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-â1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73 percent VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-â1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-â1. However, ED5 has no effect on VSMC apoptosis.
Assuntos
Animais , Ratos , Proliferação de Células , Ciclina D1/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Músculo Liso Vascular/citologia , Fator de Crescimento Transformador beta1/metabolismo , Apoptose/fisiologia , Western Blotting , Domínio Catalítico/fisiologia , Ciclina D1/fisiologia , DNA , Regulação para Baixo/fisiologia , Hiperplasia/prevenção & controle , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/patologiaRESUMO
We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-beta1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 +/- 1.52 and 72 +/- 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-beta1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-beta1. However, ED5 has no effect on VSMC apoptosis.
Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Músculo Liso Vascular/citologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Domínio Catalítico/fisiologia , Ciclina D1/fisiologia , DNA/biossíntese , Regulação para Baixo/fisiologia , Hiperplasia/prevenção & controle , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túnica Íntima/patologiaRESUMO
BACKGROUND: The Structural Descriptor Database (SDDB) is a web-based tool that predicts the function of proteins and functional site positions based on the structural properties of related protein families. Structural alignments and functional residues of a known protein set (defined as the training set) are used to build special Hidden Markov Models (HMM) called HMM descriptors. SDDB uses previously calculated and stored HMM descriptors for predicting active sites, binding residues, and protein function. The database integrates biologically relevant data filtered from several databases such as PDB, PDBSUM, CSA and SCOP. It accepts queries in fasta format and predicts functional residue positions, protein-ligand interactions, and protein function, based on the SCOP database. RESULTS: To assess the SDDB performance, we used different data sets. The Trypsion-like Serine protease data set assessed how well SDDB predicts functional sites when curated data is available. The SCOP family data set was used to analyze SDDB performance by using training data extracted from PDBSUM (binding sites) and from CSA (active sites). The ATP-binding experiment was used to compare our approach with the most current method. For all evaluations, significant improvements were obtained with SDDB. CONCLUSION: SDDB performed better when trusty training data was available. SDDB worked better in predicting active sites rather than binding sites because the former are more conserved than the latter. Nevertheless, by using our prediction method we obtained results with precision above 70%.
Assuntos
Armazenamento e Recuperação da Informação/métodos , Proteínas/química , Proteínas/fisiologia , Proteômica/métodos , Análise de Sequência de Proteína/métodos , Software , Algoritmos , Inteligência Artificial , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Bases de Dados de Proteínas , Internet , Reconhecimento Automatizado de Padrão , Conformação Proteica , Proteínas/genética , Relação Estrutura-AtividadeRESUMO
Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.
Assuntos
Peróxidos/química , Peróxidos/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Catálise , Domínio Catalítico/fisiologia , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Cinética , Metaloproteínas/química , Metaloproteínas/metabolismo , Oxirredução , Estrutura Quaternária de Proteína/fisiologia , Especificidade por Substrato/fisiologia , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismoRESUMO
Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2alpha or CK2alpha') and regulatory (CK2beta) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2alpha or CK2alpha') and the CK2beta regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2alpha that eliminate phosphorylating activity [CK2alpha(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2alpha(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.
Assuntos
Caseína Quinase II/metabolismo , Domínio Catalítico/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Western Blotting , Caseína Quinase II/genética , Células Cultivadas , DNA Complementar/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoprecipitação , Cinética , Mutação/genética , Transporte Proteico/fisiologia , TransfecçãoRESUMO
Snake venom metalloproteases (SVMPs) are a set of interesting enzymes that are one of the major components of venom affecting hemostasis. A great challenge since their discovery has been to find molecular features responsible for their hemorrhagic potency and many attempts have been made without any consistent result. Here we describe a series of comparisons between the catalytic domains of hemorrhagic and non-hemorrhagic SVMPs made with the help of bioinformatics. These involved sequence and structure-based multiple alignments, phylogenetic reconstruction, predicted physical and chemical properties, motif scanning and structural analyses. Although hemorrhagic activity seems to be complex, involving multiple factors, we found some molecular characteristics that may influence the toxic effects. Among these findings, it was possible to use a molecular surface feature to subdivide the P-I class in hemorrhagic and non-hemorrhagic SVMPs. It was also possible to suggest a role for the conserved Asp148 and Ser176 residues in the stabilization of the active site.