RESUMEN
Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase that oxidizes isomeric forms of ß-NAD(P)H. Extracellular renalase lacking its N-terminal peptide and cofactor FAD exerts various protective effects via non-catalytic mechanisms. Certain experimental evidence exists in the literature that the RP220 peptide (a 20-mer peptide corresponding to the amino acid sequence RNLS 220-239) reproduces a number of non-catalytic effects of this protein, acting on receptor proteins of the plasma membrane. The possibility of interaction of this peptide with intracellular proteins has not been studied. Taking into consideration the known role of RNLS as a possible antihypertensive factor, the aim of this study was to perform proteomic profiling of the kidneys of normotensive and hypertensive rats using RP220 as an affinity ligand. Proteomic (semi-quantitative) identification revealed changes in the relative content of about 200 individual proteins in the kidneys of hypertensive rats bound to the affinity sorbent as compared to the kidneys of normotensive animals. Increased binding of SHR renal proteins to RP220 over the normotensive control was found for proteins involved in the development of cardiovascular pathology. Decreased binding of the kidney proteins from hypertensive animals to RP220 was noted for components of the ubiquitin-proteasome system, ribosomes, and cytoskeleton.
Asunto(s)
Hipertensión , Riñón , Monoaminooxidasa , Proteómica , Ratas Endogámicas SHR , Animales , Ratas , Riñón/metabolismo , Hipertensión/metabolismo , Proteómica/métodos , Monoaminooxidasa/metabolismo , Masculino , Ligandos , Péptidos/metabolismo , Péptidos/química , Proteoma/metabolismoRESUMEN
Comparative proteomic analysis of kidney tissue from normotensive (WKY) and spontaneously hypertensive (SHR) rats revealed quantitative and qualitative changes in renal proteins. The number of renal proteins specific for WKY rats (blood pressure 110-120 mm Hg) was 13-16. There were 20-24 renal proteins specific for SHR (blood pressure 180 mm Hg and more). The total number of identified renal proteins common for both rat strains included 972-975 proteins. A pairwise comparison of all possible (SHR-WKY) variants identified 8 proteins specific only for normotensive (WKY) animals, and 7 proteins specific only for hypertensive ones (SHR). Taking into consideration their biological roles, the lack of some enzyme proteins in hypertensive rats (for example, biliverdin reductase A) reduces the production of molecules exhibiting antihypertensive properties, while the appearance of others (e.g. betaine-homocysteine S-methyltransferase 2, septin 2, etc.) can be interpreted as a compensatory reaction. Renal proteins with altered relative content (with more than 2.5-fold change) accounted for no more than 5% of all identified proteins. Among the proteins with an increased relative content in hypertensive animals, the largest group consisted of proteins involved in the processes of energy generation and carbohydrate metabolism, as well as antioxidant and protective proteins. In the context of the development of hypertension, the identified relative changes can apparently be considered compensatory. Among the proteins with the most pronounced decrease in the relative content in hypertensive rats, the dramatic reduction in acyl-CoA medium-chain synthetase-3 (ACSM3) appears to make an important contribution to the development of renal pathology in these animals.
Asunto(s)
Hipertensión , Riñón , Proteómica , Ratas Endogámicas SHR , Animales , Ratas , Hipertensión/metabolismo , Riñón/metabolismo , Proteómica/métodos , Masculino , Ratas Endogámicas WKY , Proteoma/metabolismo , Proteoma/análisis , Presión SanguíneaRESUMEN
Isatin (indoldione-2,3) is an endogenous biological regulator found in the brain, peripheral tissues, and biological fluids of humans and animals. Its biological activity is realized via isatin-binding proteins, many of which were identified during proteomic profiling of the brain of mice and rats. A number of these proteins are related to the development of neurodegenerative diseases. Previously, using a model of experimental Parkinsonism induced by a seven-day course of rotenone injections, we have observed behavioral disturbances, as well as changes in the profile and relative content of brain isatin-binding proteins. In this study, we have investigated behavioral responses and the relative content of brain isatin-binding proteins in rats with rotenone-induced Parkinsonism 5 days after the last administration of this neurotoxin. Despite the elimination of rotenone, animals exhibited motor and coordination impairments. Proteomic profiling of isatin-binding proteins revealed changes in the relative content of 120 proteins (the relative content of 83 proteins increased and that of 37 proteins decreased). Comparison of isatin-binding proteins characterized by the changes in the relative content observed in the brain right after the last injection of rotenone (n=16) and 5 days later (n=11) revealed only two common proteins (glyceraldehyde-3-phosphate dehydrogenase and subunit B of V-type proton ATPase). However, most of these proteins are associated with neurodegeneration, including Parkinson's and Alzheimer's diseases.
Asunto(s)
Isatina , Trastornos Parkinsonianos , Humanos , Animales , Ratas , Proteínas Portadoras , Isatina/farmacología , Rotenona/farmacología , Proteómica , Encéfalo , Trastornos Parkinsonianos/inducido químicamenteRESUMEN
Renalase (RNLS) is a secretory protein discovered in 2005. It plays an important role in the regulation of blood pressure. Studies by two independent laboratories have shown that administration of purified recombinant RNLS reduced blood pressure in experimental animals. However, the mechanisms of the antihypertensive effect of RNLS still remain unclear, especially in the context of the shift in the catalytic paradigm of this protein. In addition, there is growing evidence that endogenous plasma/serum RNLS, detected by enzyme immunoassay, is not an intact protein secreted into the extracellular space, and exogenous recombinant RNLS is effectively cleaved during short-term incubation with human plasma samples. This suggests that the antihypertensive effect of RNLS may be due to peptides formed during proteolytic processing. Based on the results of a bioinformatics analysis of potential RNLS cleavage sites (Fedchenko et al., Medical Hypotheses, 2022; DOI: 10.1016/j.mehy.2022.110895), a number of short peptides have been identified in the RNLS sequence that show similarity to fragments of known peptide inhibitors of angiotensin-converting enzyme. Some of them were found as a part of larger RNLS peptides, formed during RNLS cleavage by chymotrypsin and, and to a lesser extent, by trypsin.
Asunto(s)
Antihipertensivos , Monoaminooxidasa , Humanos , Secuencia de Aminoácidos , Fragmentos de Péptidos , PéptidosRESUMEN
Effects of the endogenous neuroprotector isatin and the pharmacological drug afobazole (exhibiting neuroprotective properties) on behavioral reactions and quantitative changes in the brain proteomic profile have been investigated in rats with experimental rotenone Parkinsonism. A single dose of isatin (100 mg/kg subcutaneously on the last day of a 7-day course of rotenone administration) improved the motor activity of rats with rotenone-induced Parkinsonism in the open field test (horizontal movements) and the rotating rod test. Afobazole (10 mg/kg intraperitoneally, daily during the 7-day course of rotenone administration) reduced the manifestations of rigidity and postural instability. Proteomic analysis, performed using brain samples obtained the day after the last administration of rotenone and neuroprotectors, revealed similar quantitative changes in the brain of rats with rotenone Parkinsonism. An increase in the relative content of 65 proteins and a decrease in the relative content of 21 proteins were detected. The most pronounced changes - an almost ninety-fold increase in the alpha-synuclein content - were found in the brains of rats treated with isatin. In animals of the experimental groups treated with "Rotenone + Isatin", as well as "Rotenone + Afobazole", the increase in the relative content of this protein in the brain was almost 60 and 50 times higher than the control values. Taking into consideration the known data on the physiological role of alpha-synuclein, an increase in the content of this protein in the brain upon administration of neuroprotectors to animals with rotenone Parkinsonism may represent a compensatory reaction, at least in the early stages of this disease and the beginning of its treatment.
Asunto(s)
Isatina , Fármacos Neuroprotectores , Trastornos Parkinsonianos , Ratas , Animales , Rotenona/efectos adversos , Rotenona/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Isatina/farmacología , Isatina/metabolismo , Octoxinol/efectos adversos , Octoxinol/metabolismo , alfa-Sinucleína , Proteómica , Encéfalo , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismoRESUMEN
Renalase (RNLS) is a recently discovered protein, which plays different roles inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5), while extracellular RNLS lacks its N-terminal peptide, FAD cofactor, and exhibits various protective effects in a non-catalytic manner. Certain evidence exists, that plasma/serum RNLS is not an intact protein secreted into the extracellular space, and exogenous recombinant RNLS is effectively degraded during short-term incubation with human plasma samples. Some synthetic analogues of the RNLS sequence (e.g. the Desir's peptide RP-220, a 20-mer peptide corresponding to the RNLS sequence 220-239) have effects on cell survival. This suggests that RNLS-derived peptides, formed during proteolytic processing, may have own biological activity. Based on results of a recent bioinformatics analysis of potential cleavage sites of RNLS (Fedchenko et al., Medical Hypotheses, 2022) we have investigated the effect of four RNLS-derived peptides as well as RP-220 and its fragment (RP-224) on the viability of two cancer cell lines: HepG2 (human hepatoma) and PC3 (prostate cancer). Two RNLS-derived peptides (RP-207 and RP-220) decreased the viability of HepG2 cells in a concentration dependent manner. The most pronounced and statistically significant effect (30-40% inhibition of cell growth) was observed at 50 µM concentration of each peptide. In the experiments with PC3 cells five of six RNLS-derived peptides had a significant impact on the cell viability. RP-220 and RP-224 decreased cell viability; however, no concentration dependence of this effect was observed in the range of concentrations studied (1-50 µM). Three other RNLS-derived peptides (RP-207, RP-233, and RP-265) increased viability of PC3 cells by 20-30%, but no concentration-dependence of this effect was found. Data obtained suggest that some RNLS-derived peptides may influence the viability of various cells and manifestation and direction of the effect (increase of decrease of the cell viability) is cell-type-specific.
Asunto(s)
Monoaminooxidasa , Péptidos , Humanos , Masculino , Células PC-3 , Péptidos/farmacología , Línea CelularRESUMEN
The neurotoxins rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (ÐPTP) are used for modeling Parkinson's disease in animals (PD). They induce the mitochondrial respiratory chain dysfunction, which leads to the dopaminergic (DA) neuron degeneration. The advantage of the rotenone model consists in ability of rotenone to cause neurodegeneration showing symptoms and molecular biological characteristics similar to those of PD. Isatin (indoldione-2,3) is an endogenous regulator found in tissues and biological fluids of humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. In this work we have investigated behavioral reactions and profiles of brain isatin-binding proteins of rats with Parkinson's syndrome (PS) in comparison with the corresponding parameters of MPTP-induced Parkinsonism in mice. Systemic injection of rotenone caused severe PS comparable with the effect of MPTP injection. It was accompanied by significant body weight loss, death, oligokinesia, muscular rigidity, and postural instability of animals. In spite of the same pathogenic basis of PS caused by rotenone and MPTP, the molecular mechanisms of their action differ. In the case of rotenone-induced PS, the pool of isatin-binding proteins common of the control rats and the rats with PS (146) significantly exceeded the pool of the common proteins of control mice and mice with PS induced by MPTP, whether right after neurotoxin injection (27), or (all the more) in a week after the MPTP injection (14). The comparison of isatin-binding proteins specific of the animals with MPTP-induced PS and with the rotenone-induced PS (as compared with the control animals) revealed total absence of proteins common of these two models of PD. It is to be noted that both neurotoxins particularly affected the proteins participating in the signal transmission and enzyme activity regulation. The changes of the profile of isatin-binding proteins in response to the injection of rotenone suggest that the neuroprotector isatin could also influence positively in the case of the rotenone model of PD.
Asunto(s)
Isatina , Trastornos Parkinsonianos , Animales , Ratones , Ratas , Encéfalo , Proteínas Portadoras , Neurotoxinas , RotenonaRESUMEN
Isatin (indole-2,3-dione) is an endogenous regulator exhibiting various effects mediated by numerous isatin-binding proteins localized in different compartments of cells of the brain and peripheral tissues. It attenuates manifestations of experimental parkinsonism induced by administration of the MPTP neurotoxin and reduces the movement disorders characteristic of this disease. The molecular mechanisms of the neuroprotective action of isatin include its direct interaction with proteasomes, intracellular supramolecular complexes responsible for the targeted elimination of proteins. Incubation of fractions of 26S and 20S rabbit brain proteasomes, containing the whole spectrum of proteasomal subunits, as well as a number of proteasome-associated proteins, with isatin (100 µM) had a significant impact on the profile of released proteins. In the case of 26S proteasomes containing, in addition to the core part (20S proteasome), 19S regulatory subparticles, incubation with isatin resulted in a more than threefold increase in the number of dissociated proteins. In the case of 20S proteasomes (containing only the 20S core particle), incubation with isatin resulted in a significant decrease in the number of dissociated proteins compared to the control. Our results indicate an important role of the regulatory 19S subunit components in the formation of the proteasome subproteome and the sensitivity of these supramolecular complexes to isatin.
Asunto(s)
Isatina , Trastornos Parkinsonianos , Animales , Encéfalo/metabolismo , Isatina/metabolismo , Isatina/farmacología , Trastornos Parkinsonianos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas , ConejosRESUMEN
We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteínas Ubiquitinadas , Animales , Encéfalo/metabolismo , Hígado/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Conejos , Ubiquitina/metabolismoRESUMEN
Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.
Asunto(s)
Isatina , Neurotoxinas , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Encéfalo/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Isatina/metabolismo , Isatina/farmacología , Ratones , Mitocondrias/metabolismo , Neurotoxinas/metabolismo , Neurotoxinas/farmacología , ProteómicaRESUMEN
Good evidence exists that the ubiquitin-proteasome system (UPS) plays an important role in degradation of mitochondrial proteins and membrane proteins associated with mitochondria (MAM proteins). Mitochondria contain all components of the ubiquitin-conjugating system, which are necessary for the attachment of ubiquitin molecules to target proteins, subjected to subsequent degradation in proteasomes. An important stage in the delivery of proteins for proteolytic degradation in proteasomes is their interaction with ubiquitin receptors located on the regulatory subunit (19S) of the proteasome: the Rpn10 or Rpn13 subunit. These subunits make basically the same contribution to the subsequent translocation of target proteins to the core part of the proteasome. A comparative study of mouse brain mitochondrial subproteomes bound to Rpn10 and Rpn13 subunits revealed a high specificity of the repertoire of Rpn10 and Rpn13-binding proteins. Moreover, proteins, for which mitochondrial localization or association with mitochondrial membranes was previously shown, prevailed in the case of using the Rpn13 subunit as an affinity ligand (Rpn13-binding proteins). This suggests that Rpn10 and Rpn13 play different roles in the degradation of mitochondrial proteins and MAM.
Asunto(s)
Encéfalo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma , Proteínas de Unión al ARN/metabolismo , Animales , RatonesRESUMEN
The review summarizes the data of our research and published studies on the ubiquitination of brain mitochondrial proteins and its changes during the development of experimental parkinsonism and administration of the neuroprotector isatin (indole-2,3-dione) with special attention to the mitochondrial ubiquitin-conjugating system and location of ubiquitinated proteins in these organelles. Incubation of brain mitochondrial fraction with biotinylated ubiquitin in vitro resulted in the incorporation of biotinylated ubiquitin in both mitochondrial and mitochondria-associated proteins. According to the interactome analysis, the identified non-ubiquitinated proteins are able to form tight complexes with ubiquitinated proteins or their partners and components of mitochondrial membranes, in which interactions of ubiquitin chains with the ubiquitin-binding protein domains play an important role. The studies of endogenous ubiquitination in the total brain mitochondrial fraction of C57Bl mice performed in different laboratories have shown that mitochondrial proteins represent about 30% of all ubiquitinated proteins. However, comparison of brain subproteomes of mitochondrial ubiquitinated proteins reported in the literature revealed significant differences both in their composition and involvement of identified ubiquitinated proteins in biological processes listed in the Gene Ontology database. The development of experimental parkinsonism in C57Bl mice induced by a single-dose administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a decrease in the total number of mitochondrial ubiquitinated proteins and increase in the number of oxidized mitochondrial proteins containing the ubiquitin signature (K-ε-GG). Comparison of ubiquitinated proteins associated with the mouse brain mitochondrial fraction and mouse brain mitochondrial proteins bound to the proteasome ubiquitin receptor (Rpn10 subunit) did not reveal any common proteins. This suggests that ubiquitination of brain mitochondrial proteins is not directly related to their degradation in the proteasomes. Proteomic profiling of brain isatin-binding proteins identified enzymes involved in the ubiquitin-conjugating system functioning. Mapping of the identified isatin-binding proteins to known metabolic pathways indicates their participation in the parkin (E3 ubiquitin ligase)-associated pathway (CH000000947). The functional links involving brain mitochondrial ubiquitinated proteins were found only in the group of animals with the MPTP-induced parkinsonism, but not in animals treated with MPTP/isatin or isatin only. This suggests that the neuroprotective effect of isatin may be associated with the impaired functional relationships of proteins targeted to subsequent degradation.
Asunto(s)
Encéfalo/metabolismo , Trastornos Parkinsonianos/patología , Ubiquitina/metabolismo , Animales , Autofagia , Redes y Vías Metabólicas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/veterinaria , Complejo de la Endopetidasa Proteasomal/metabolismo , UbiquitinaciónRESUMEN
Isatin (indol-2,3-dione), an endogenous biofactor found in the brain, peripheral tissues and biological body fluids of humans and animals, exhibits a wide range of biological and pharmacological activities. They are realized via interaction with numerous isatin-binding proteins. Some of these proteins identified during proteomic profiling of the brain are involved in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl (selegiline), a pharmacological agent used for treatment of Parkinson's disease. In this study, we have investigated the effect of a single dose administration of isatin (100 mg/kg) and deprenyl (10 mg/kg) to mice on the profile of the brain isatin-binding proteins. Comparative proteomic analysis of brain isatin-binding proteins of mice treated with isatin or deprenyl resulted in identification of a representative group of proteins (n=200) sensitive to the administration of these substances. The change in the profile of isatin-binding proteins may be obviously attributed to accumulation of isatin and deprenyl in the brain and their interaction with target proteins; this prevents protein binding to the affinity sorbent. Thus identified brain isatin-binding proteins of the control animals obviously represent specific targets that interact directly with isatin (and also with deprenyl) in vivo. Isatin or deprenyl administered to animals interact with these proteins and thus inhibit their binding to the affinity sorbent (immobilized isatin analogue).
Asunto(s)
Encéfalo/efectos de los fármacos , Proteínas Portadoras/metabolismo , Isatina/farmacología , Fármacos Neuroprotectores/farmacología , Selegilina/farmacología , Animales , Encéfalo/metabolismo , Ratones , ProteómicaRESUMEN
It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.
Asunto(s)
Técnicas Biosensibles , Complejo de la Endopetidasa Proteasomal/química , Proteínas Ubiquitinadas/química , Humanos , Ubiquitina , UbiquitinaciónRESUMEN
Amyloid-ß peptide (1-42) (Aß1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aß1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aß1-42. It is reasonable to expect that dissociation of the Aß1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aß1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aß1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aß-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aß1-42 and its fragments (Aß1-28, Aß12-28, Aß25-35). Aß1-42 and its fragments (Aß1-28, Aß12-28, Aß25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aß1-42 and Aß25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aß peptides except Aß1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aß1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aß12-28 and Aß25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aß studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aß1-42, and its aggressive truncated peptides (Aß25-35).
Asunto(s)
Isatina/metabolismo , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Gliceraldehído-3-Fosfato Deshidrogenasas , Humanos , Fragmentos de PéptidosRESUMEN
Isatin (indol-2,3-dione) is an endogenous indole found in the brain, peripheral tissues and biological body fluids of humans and animals. Its wide spectrum of biological activity is realized via interaction with numerous isatin-binding proteins; these include proteins playing an important role in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl, a pharmacological agent used for treatment of Parkinson's disease. In this study, the effects of the course of deprenyl (1 mg/kg) and isatin (20 mg/kg) administration for 21 days on the profile of the isatin-binding proteins of the liver of mice have been investigated. Proteomic profiling of liver isatin-binding proteins of control mice by means of 5-aminocaproylisatin as an affinity ligand resulted in identification of 105 proteins. Treatment of animals with a low dose of isatin slightly decreased (up to 91), while injections of deprenyl slightly increased (up to 120) the total number of isatin-binding proteins. 75 proteins were common for all three groups; they represented from 62.5% (in deprenyl treated mice) and 71% (in control mice), to 82% (isatin treated mice) of the total number of identified liver isatin-binding proteins. Proteomic analysis of the isatin-binding proteins of mice treated with isatin (20 mg/kg) or deprenyl (1 mg/kg) for 21 days revealed a representative group of proteins (n=30) that were sensitive to the administration of these substances. Taking into account the previously obtained results, it is reasonable to suggest that the change in the profile of isatin-binding proteins may be attributed to accumulation of isatin and deprenyl in the liver and interaction with target proteins prevents their subsequent binding to the affinity sorbent. In this context, the identified isatin-binding liver proteins of control animals that do not bind to the affinity sorbent (immobilized isatin analogue) after treatment of animals with either deprenyl or isatin appear to be specific targets directly interacting with isatin in vivo.
Asunto(s)
Isatina/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Fármacos Neuroprotectores/farmacología , Selegilina/farmacología , Animales , Unión Competitiva , Isatina/administración & dosificación , Isatina/metabolismo , Ligandos , Masculino , Metaboloma/efectos de los fármacos , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/metabolismo , Unión Proteica , Proteómica , Selegilina/administración & dosificación , Selegilina/metabolismoRESUMEN
Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: the ATP-dependent addition of (typically four sequential) residues of the low-molecular ubiquitin protein, involving the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme and ubiquitin ligase. The cytoplasm and nucleoplasm proteins labeled in this way are then digested in 26S proteasomes. However, in recent years it has become increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent protein degradation in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate further increase. Since 26S of proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles and whole cells, has the most serious consequences for the whole organism.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/química , Proteolisis , Animales , Humanos , Ubiquitina , UbiquitinaciónRESUMEN
It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass-spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.
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Complejos Multiproteicos/química , Biosíntesis de Proteínas , Proteómica , Humanos , Hígado , Espectrometría de Masas , Peso MolecularRESUMEN
RATIONALE: Renalase is a recently discovered kidney secretory protein, which is considered as an important component involved in blood pressure regulation. Although altered levels of renalase have been detected in plasma and urine of patients with various kidney diseases, there is certain inconsistency of changes in the renalase levels reported by different laboratories. The latter is obviously associated with the use of the ELISA as the only available approach for quantitative analysis of renalase. Thus there is a clear need for the development of antibody-independent approaches for renalase quantification. METHODS: We have developed a new method for quantitative determination of human renalase, which is based on mass spectrometric detection of a proteotypic peptide containing С-terminal 13 C15 N-labelled lysine. It corresponds to a tryptic peptide of human renalase, which has been previously detected in most mass spectrometric determinations of this protein. RESULTS: Using the labelled peptide H-EGDCNFVAPQGISSIIK-OH, corresponding to positions 100-116 of the human renalase sequence, as an internal standard and recombinant human renalase we have generated a calibration curve, which covered the concentration range 0.005-50 ng/mL with a limit of quantitation of 5 pg/mL. Using this calibration curve we were able to detect urinary renalase only after enrichment of initial urinary samples by ammonium sulfate precipitation (but not in untreated urine). CONCLUSIONS: Results of our study indicate that quantitative determination of renalase based on mass spectrometric detection of a proteotypic peptide labelled with stable isotopes gives significantly lower values of this protein in human urine than those reported in the literature and based on the ELISA.
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Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Monoaminooxidasa/orina , Adulto , Anciano , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Monoaminooxidasa/metabolismo , Adulto JovenRESUMEN
Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this work, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase: five isoforms of cytochromes P450, two isoforms of cytochrome b5, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown that isatin binds specifically only to cytochromes P450 with high affinity (the equilibrium dissociation constant (Kd) is about 10-8 M).