[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.

[Unique structural organization of ATP-dependent LonA proteases].

Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, regions that are capable to include intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA+ proteins, because in addition to the classic AAA+ module they contain a part of another AAA+ module, namely the alpha-helical domain including a coiled-coil region, which is similar to the alpha-helical domain of the AAA(+)-1 module of the chaperone-disagregases ClpB/Hsp104.

[Selective inhibitors of plasmepsin II from Plasmodium falciparum based on pepstatin].

A number of new inhibitors of plasmepsin II (PlmII) from Plasmodium falciparum, one of the key factors of malarial parasite survival, were synthesized. The inhibitors are analogues of pepstatin with various variants of Ala residue substitutions. Effects of the inhibitors on human PlmII and cathepsin D were studied. Inhibition of PlmII by the substrate was found, which required the use of the modified Henderson method for the determination of inhibition constants. Two synthesized inhibitors were shown to exhibit a pronounced selectivity to PlmII (K(i) = 5.5 and 5 nM) in comparison with cathepsin D (K(i) = 230 and 3000 nM, respectively).

New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

[5-fluoro-tryptophan-containing N-terminal domain of the alpha-subunit of the Torpedo californica acetylcholine receptor: preparation in E. coli and 19F NMR study]. / Poluchenie v E. coli i 19F-IaMR issledovanei 5-ftortriptofansoderzhashchego n-kontsevogo domena alpha-sub''edinitsy atsetilcholinovogo retseptora Torpedo californica.

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

[Ulysses retrotransposon aspartate proteinase (Drosophila virilis)]. / Aspartatnaia proteinaza retrotranspozona Ulysses (Drosophila virilis).

Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity.

Selective protein degradation is an energy-dependent process performed by high-molecular-weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX-KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis.

[Synthesis and characterisation of ATP-dependent forms of Lon-proteinase with modified N-terminal domain from Escherichia coli]. / Polucheniie i issledovanie modifitsirovannykh v oblasti N-kontsevogo domena form ATP-zavisimoi Lon-proteinazy iz Escherichia coli.

The functional domain boundaries of the ATP-dependent Lon proteases were identified by comparative analysis of the amino acid sequences of the enzymes from evolutionarily distant organisms. Modified forms of the Escherichia coli Lon protease with the elongated or substituted N-terminal domain and a truncated enzyme lacking the N-terminal domain were obtained through genetic engineering methods. Analysis of the enzymatic properties of the resulting modified forms of Lon protease revealed the importance of the N-terminal domain in its function.

HIV-I protease. Cloning, expression, and purification.

A new method for obtaining HIV-I protease was suggested. Fusion proteins composed of the N-terminal fragment of human gamma-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed in Escherichia coli cells. The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I was cleaved by enterokinase. The solubility of protein I was increased by treating with Na-sulfite/Na-tetrathionate under denaturing conditions. Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found. The hydrolysis products were separated by reversed-phase FPLC. The amount of tryptophan and cysteine residues in the enzyme obtained was estimated. The activity of HIV-I protease was determined using the chromogenic peptide. AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of beta-galactosidase and a fragment of gag proteins, including p17-p24 processing site.