Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X-100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin-binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5-aminocarproyl-isatin, and 5-aminoisatin as the affinity ligands. The K(d) values (of 0.1-20 microM) obtained during the optical biosensor experiments were consistent with the range of K(d) values recently reported for [(3)H]isatin binding to brain sections. Although the number of isatin-binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one-third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.
Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [(3)H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real-time beta-imager, [(3)H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5-aminocaproyl-isatin-Sepharose, we have investigated the distribution of [(3)H]isatin specific binding sites in the rat brain, characterized their K(d) and B(max), and identified some individual brain isatin binding proteins. The binding of [(3)H]isatin to rat brain sections was saturable and characterized by K(d) values (of 0.2-0.3 microM) consistent with physiological concentrations. The highest B(max) was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [(3)H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X-100 lysates of whole-brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde-3-phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton-related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.
Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.
The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer’s disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.
BackgroundRenalase is a recently discovered secretory protein involved in regulation of arterial blood pressure in humans and animals. Results of animal experiments from independent laboratories indicate that administration of human recombinant renalase decreases blood pressure and some genetically predisposed hypertensive rats have lowered renalase levels.Material/MethodsThe levels of renalase mRNA expression in brain hemispheres, heart, and kidneys of spontaneously hypertensive rats (SHR) with moderate (140–180 mm Hg) or high (>180 mm Hg) hypertension and of control Wistar-Kyoto (WKY) rats were analyzed using real-time PCR.ResultsSpontaneously hypertensive rats with high hypertension (>180 mm Hg) had a lower renalase mRNA level in brain hemispheres, and higher heart and kidney renalase mRNA levels compared with control WKY rats. In SHR with a moderate increase in arterial blood pressure (140–180 mm Hg), the tissue renalase mRNA changed in the same direction but did not reach the level of statistical significance as compared with control rats.ConclusionsThe results indicate that the development of hypertension in SHR is accompanied by altered expression of the renalase gene in the examined organs as compared with control WKY rats. The brain and peripheral tissues renalase mRNA levels demonstrate opposite trends, which are obviously crucial for impaired regulation of blood pressure in SHR.
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