RESUMO
Kex2 is the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave at sites containing pairs of basic residues. Here, we studied the effects of KCl on the individual rate constants of association, dissociation, acylation and deacylation and determined the thermodynamic parameters at each step of the Kex2 reaction. Potassium bound Kex2 with KD=20.3mM. The order in which potassium entered the reaction system modified the effect of activation or inhibition, which depended on the size of the substrate. A possible allosteric potassium binding site at the S6 subsite was involved in activation, and a distant site located between the catalytic domain and the P-domain was involved in inhibition. Potassium decreased the energetic barriers of almost all steps of catalysis. The acylation of Ac-PMYKR-AMC in the absence of potassium was the rate-limiting step. Therefore, for substrates containing a P1-Arg, the deacylation step is not necessarily the rate-limiting event, and other residues at the P' positions may participate in controlling the acylation and deacylation steps. Thus, it is reasonable to conclude that potassium is involved in the processing of the α-mating factor that promotes Ca2+ mobilization by activating a high-affinity Ca2+-influx system to increase the cytosolic [Ca2+], resulting in the activation of channels that are essential for the survival of Saccharomyces cerevisiae cells.
Assuntos
Potássio/farmacologia , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Acilação , Cálcio/metabolismo , Potássio/química , Especificidade por SubstratoRESUMO
Kex2 is a Ca2+-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P1' is affected by the type of residue in the P3 position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KM values for peptides with P2' substitutions containing Tyr in P3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcat and KM, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism.
Assuntos
Fator de Acasalamento/metabolismo , Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico , Concentração de Íons de Hidrogênio , Cinética , Fator de Acasalamento/genética , Modelos Moleculares , Peptídeos/genética , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.
Assuntos
Fator VIII/química , Fator VIII/metabolismo , Furina/metabolismo , Animais , Células CHO , Cricetulus , Fator VIII/genética , Humanos , Pró-Proteína Convertases/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismoRESUMO
Parkinson's disease (PD) is a multifactorial neurodegenerative disorder, characterised by the progressive loss of midbrain dopaminergic neurons and a variety of motor symptoms. The gene coding for the phospholipid phosphatase 3, PLPP3 (formerly PPAP2B or LPP3), maps within the PARK10 locus, a region that has been linked with increased risk to late-onset PD. PLPP3 modulates the levels of a range of bioactive lipids controlling fundamental cellular processes within the central nervous system. Here we show that PLPP3 is enriched in astroglial cells of the adult murine ventral midbrain. Conditional inactivation of Plpp3 using a Nestin::Cre driver results in reduced mesencephalic levels of sphingosine-1-phosphate receptor 1 (S1P1), a well-known mediator of pro-survival responses. Yet, adult PLPP3-deficient mice exhibited no alterations in the number of dopaminergic neurons or in the basal levels of striatal extracellular dopamine (DA). Potassium-evoked DA overflow in the striatum, however, was significantly decreased in mutant mice. Locomotor evaluation revealed that, although PLPP3-deficient mice exhibit motor impairment, this is not progressive or responsive to acute L-DOPA therapy. These findings suggest that disruption of Plpp3 during early neural development leads to dopaminergic transmission deficits in the absence of nigrostriatal degeneration, and without causing an age-related locomotor decline consistent with PD.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Loci Gênicos , Locomoção/fisiologia , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas de Transferência de Fosfolipídeos/deficiência , Proteínas de Transferência de Fosfolipídeos/genética , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Transdução de SinaisRESUMO
Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/ß-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/ß-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/ß-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive ß-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype-phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.
Assuntos
Estudos de Associação Genética , Mutação , Pró-Proteína Convertases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Serina Endopeptidases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Masculino , Pró-Proteína Convertases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteólise , Serina Endopeptidases/genética , Transferases (Outros Grupos de Fosfato Substituídos)/químicaRESUMO
BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) has a key role in the regulation of plasma low-density lipoprotein (LDL) cholesterol by enhancing the degradation of LDL receptor. Functional variants in PCSK9 have been associated with differences in plasma lipids and may contribute to the variability of the response to cholesterol-lowering drugs. OBJECTIVE: To investigate the influence of PCSK9 variants on plasma lipid profile and response to atorvastatin in Brazilian subjects. METHODS: PCSK9 E670G, I474V, and R46L single nucleotide polymorphisms (SNPs) and plasma lipids were evaluated in 163 hypercholesterolemics (HC) and 171 normolipidemics (NL). HC patients with indication for cholesterol-lowering drug therapy (n = 128) were treated with atorvastatin (10 mg/d/4 wk). PCSK9 SNPs were analyzed by real time polymerase chain reaction. RESULTS: Frequencies of the PCSK9 SNPs were similar between the HC and NL groups. Logistic regression analysis showed a trend of association between PCSK9 E670G and hypercholesterolemia after adjustment for covariates (P = .059). The 670G allele was associated with high basal levels of LDL cholesterol (P = .03) in HC patients using the extreme discordant phenotype method. No association tests were performed for R46L variant because of its very low frequency, whereas the I474V polymorphism and PCSK9 haplotypes were not related to hypercholesterolemia or variability on plasma lipids in both NL and HC groups (P > .05). LDL cholesterol reduction in response to atorvastatin was not influenced by PCSK9 genotypes or haplotypes. CONCLUSIONS: PCSK9 E670G polymorphism but not I474V contributes to the variability on plasma LDL cholesterol levels in hypercholesterolemic subjects. Both PCSK9 variants have no influence on cholesterol-lowering response to atorvastatin.
Assuntos
Biomarcadores Farmacológicos/metabolismo , LDL-Colesterol/metabolismo , Hipercolesterolemia/genética , Mutação/genética , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Adulto , Idoso , Atorvastatina , Brasil , Feminino , Ácidos Heptanoicos/administração & dosagem , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Pirróis/administração & dosagem , Serina Endopeptidases/metabolismo , Resultado do TratamentoAssuntos
Aterosclerose/terapia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Pró-Proteína Convertases/metabolismo , Pró-Proteína Convertases/uso terapêutico , Serina Endopeptidases/metabolismo , Serina Endopeptidases/uso terapêutico , LDL-Colesterol/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Polimorfismo Genético , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/farmacologia , Fatores de Risco , Serina Endopeptidases/farmacologiaAssuntos
Humanos , Aterosclerose/terapia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Pró-Proteína Convertases/metabolismo , Pró-Proteína Convertases/uso terapêutico , Serina Endopeptidases/metabolismo , Serina Endopeptidases/uso terapêutico , LDL-Colesterol/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Polimorfismo Genético , Pró-Proteína Convertases/farmacologia , Fatores de Risco , Serina Endopeptidases/farmacologiaRESUMO
Here we report a detailed analysis of magnesium (Mg²+) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg²+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR↓FAGV-Q-EDDnp (from measles virus fusion protein F0 and Abz-RERRRKKR↓GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl2. It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg²+ is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg²+ ions, which bind to furin with a K(d) value of 1.1 mM.