A Comparative Kidney Transcriptome Analysis of Bicarbonate-Loaded insrr-Null Mice.

The maintenance of plasma pH is critical for life in all organisms. The kidney plays a critical role in acid-base regulation in vertebrates by controlling the plasma concentration of bicarbonate. The receptor tyrosine kinase IRR (insulin receptor-related receptor) is expressed in renal ß-intercalated cells and is involved in alkali sensing due to its ability to autophosphorylate under alkalization of extracellular medium (pH > 7.9). In mice with a knockout of the insrr gene, which encodes for IRR, urinary bicarbonate secretion in response to alkali loading is impaired. The specific regulatory mechanisms in the kidney that are under the control of IRR remain unknown. To address this issue, we analyzed and compared the kidney transcriptomes of wild-type and insrr knockout mice under basal or bicarbonate-loaded conditions. Transcriptomic analyses revealed a differential regulation of a number of genes in the kidney. Using TaqMan real-time PCR, we confirmed different expressions of the slc26a4, rps7, slc5a2, aqp6, plcd1, gapdh, rny3, kcnk5, slc6a6 and atp6v1g3 genes in IRR knockout mice. Also, we found that the expression of the kcnk5 gene is increased in wild-type mice after bicarbonate loading but not in knockout mice. Gene set enrichment analysis between the IRR knockout and wild-type samples identified that insrr knockout causes alterations in expression of genes related mostly to the ATP metabolic and electron transport chain processes.

Changes in the Expression of the gapdh Gene in the Organs of insrr Knockout Mice.

The most important property of a living organism is the maintenance of optimal acid-base balance and the ionic composition of the internal environment. The kidneys are one of the main pH-regulating organs in the body. Receptor tyrosine kinase IRR (an insulin receptor-related receptor) is an alkaline pH-sensor. In mice (Mus Musculus) with a knockout of the insrr gene encoding the IRR receptor, bicarbonate secretion is impaired under the conditions of alkaline loading, which indicates the role of the receptor tyrosine kinase IRR in the regulation of acid-base balance in the body. In order to search for proteins functionally associated with the receptor tyrosine kinase IRR, we performed a large-scale sequencing of the mouse kidney transcriptome of wild type and insrr knockout mice kept under normal conditions and under alkaline conditions. As a result, we found a decrease in the gapdh gene expression in the kidneys of insrr knockout mice compared to wild type mice. RNA sequencing data were confirmed by TaqMan real-time PCR and Western blotting. Using the TaqMan real-time PCR method, we revealed a decrease in the level of gapdh expression not only in the kidneys, but also in the liver and brain of insrr knockout mice. Thus, the changes in the gapdh gene expression in the kidneys of insrr knockout mice may indicate a functional relationship between genes and a possible role of GAPDH in previously undescribed molecular mechanisms of regulation of acid-base balance in the body.

Role of the Inserted α-Helical Domain in E. coli ATP-Dependent Lon Protease Function.

Multidomain ATP-dependent Lon protease of E. coli (Ec-Lon) is one of the key enzymes of the quality control system of the cellular proteome. A recombinant form of Ec-Lon with deletion of the inserted characteristic α-helical HI(CC) domain (Lon-dHI(CC)) has been prepared and investigated to understand the role of this domain. A comparative study of the ATPase, proteolytic, and peptidase activities of the intact Lon protease and Lon-dHI(CC) has been carried out. The ability of the enzymes to undergo autolysis and their ability to bind DNA have been studied as well. It has been shown that the HI(CC) domain of Ec-Lon protease is required for the formation of a functionally active enzyme structure and for the implementation of protein-protein interactions.

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

Structural and functional analyses of the sixth site of neurexin alternative splicing.

In this study, we found the sixth site of alternative splicing (SS6) of neurexin 1a from the rat brain. This site is located between the fifth LNS and the third EGF-like domains. The insertion in the SS6 site corresponds to the 9-residue peptide VALMKADLQ, which is conserved among animals. We demonstrated that the SS6 insertion regulates tissue-specific expression of neurexin 1α.

[The Effect of Mutations in the Inserted Domain of ATP-Dependent Lon Protease from E. coli on the Enzyme Function].

ATP-Dependent protease LonA from E. coli (Ec-Lon), belonging to the superfamily of AAA+ proteins, is a key member of the protein quality control system in bacterial cells. Ec-Lon functions as homohexamer and degrades abnormal and defective polypeptides as well as a number of regulatory proteins by the processive mechanism. Ec-Lon subunit includes--the both ATPase and proteolytic components (AAA+ module and P domain) in addition to the unique non-catalytic region formed by the N-terminal (N) and the inserted c-helical (HI(CC)) domains. The mutant forms Lon-R164A, Lon-R192A and Lon-Y294A have been obtained and characterized in order to reveal the role of the HI (CC) domain for the enzyme functioning. C-Terminal part of the HI (CC) domain is shown to display an allosteric effect on the efficiency of the enzyme ATPase and proteolytic sites while its coiled-coil (CC) region is involved in the interaction with the protein substrate.

[Protective properties of recombinant IgA1 protease from meningococcus].

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

[Role of the α-helical domains in the functioning of ATP-dependent Lon protease of Escherichia coli].

Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA+ proteins. Their subunits are formed by five successively connected domains: N-terminal (N), α-helical (HI(CC)), nucleotide binding (NB), the second α-helical (H) and proteolytic (P). The presence of the inserted HI(CC) domain defines the uniqueness of LonA proteases among AAA+ proteins. The role of α-helical domains in the LonA protease functioning is investigated on the example of E. coli Lon protease (Ec-Lon). A comparative study of properties of the intact Ec-Lon and its mutants of Lon-R164A and Lon-R542A with the substitutions of arginine residues located in similar positions in the HI(CC) and H domains is carried out. The H domain is shown to play a crucial role for the ATP hydrolysis and enzyme binding to the target protein. HI(CC) domain does not have a fundamental significance for the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining the enzyme stability. The participation of HI(CC) domain in formation of the spatial structures of LonA proteases and/or formation of their complexes with DNA is suggested.

Deficient Response to Experimentally Induced Alkalosis in Mice with the Inactivated insrr Gene.

Currently, the molecular mechanisms of the acid-base equilibrium maintenance in the body remain poorly understood. The development of alkalosis under various pathological conditions poses an immediate threat to human life. Understanding the physiological mechanisms of alkalosis compensation may stimulate the development of new therapeutic approaches and new drugs for treatment. It was previously shown that the orphan insulin receptor-related receptor (IRR) is activated by mildly alkaline media. In this study, we analyzed mutant mice with targeted inactivation of theinsrr gene encoding IRR, and revealed their phenotype related to disorders of the acid-base equilibrium. Higher concentrations of bicarbonate and CO(2)were found in the blood ofinsrr knockout mice in response to metabolic alkalosis.

[Identification of proteins in complexes with alpha-latrotoxin receptors].

A thorough analysis of proteins capable of interacting with presynaptic receptors of alpha-latrotoxin was carried out. The protein components of receptor complexes were isolated from rat brain membranes by affinity chromatography on immobilized alpha-latrotoxin and antibodies to the cytoplasmic moiety of the calcium-independent receptor of alpha-latrotoxin (CIRL) followed by analysis by mass spectrometry. Several proteins were identified, with structural proteins, intracellular signal proteins, and proteins involved in the endocytosis and transport of synaptic vesicles being among them.