Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Truncated Variants of Serratia proteamaculans Oligopeptidase B Having Different Activities.

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75 kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeon Desulfurococcus kamchatkensis.

The crystal structure of the aminopeptidase APDkam589 from the thermophilic crenarchaeon Desulfurococcus kamchatkensis was determined at a resolution of 3.0 Å. In the crystal, the monomer of APDkam589 and its symmetry-related monomers are densely packed to form a 12-subunit complex. Single-particle electron-microscopy analysis confirms that APDkam589 is present as a compact dodecamer in solution. The APDkam589 molecule is built similarly to the molecules of the PhTET peptidases, which have the highest sequence identity to APDkam589 among known structures and were isolated from the more thermostable archaeon Pyrococcus horikoshii. A comparison of the interactions of the subunits in APDkam589 with those in PhTET1, PhTET2 and PhTET3 reveals that APDkam589 has a much lower total number of salt bridges, which correlates with the lower thermostability of APDkam589. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. A superposition of the structure of APDkam589 with those having a high sequence similarity to APDkam589 reveals that, although the positions of Trp45, Trp252 and Trp358 are not conserved in the sequences, the spatial locations of the Trp residues in these models are similar.

Identification of the ligand in the structure of the protein with unknown function STM4435 from Salmonella typhimurium.

The unidentified ligand, which is present in the crystal of the protein with unknown function STM4435 from Salmonella typhimurium, was identified using a combination of high-resolution X-ray crystallography and accurate-mass time-of-flight mass spectrometry. The identified glycerol was present as a component of the solutions used for the isolation and crystallization of the protein and serves as the ligand mimicking the natural metabolite, presumably, 2-keto-myo-isonitol, which is indicative of the involvement of STM4435 in the myo-isonitol catabolism. The results of the present study show that this approach holds promise in complex studies aimed at determining, refining, or confirming the protein functions.

[A new approach for study of structural and functional properties of proteins with unknown functions].

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.

[Amyloid-like fibrils forming and fibroblasts destruction in Tenon's capsule in progressive myopia as a result of pigment epithelium derived factor resistance to restricted proteolysis].

We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

[Haponin (eIF1AD) interacts with glyceraldehyde 3-phosphate dehydrogenase in the CHO-K1 cell line].

Haponin (HLDF-alike protein) was previously identified from the human promyelocytic leukemia HL-60 cell line. For the functional study of this protein, we obtained recombinant haponin with an N-terminal hexahistidine tag using a baculovirus expression system. Antibodies against 6xHis-haponin were produced, and the expression of endogenous haponin was demonstrated in mammalian cell lines of different origin. Using affinity chromatography and immunoprecipitation methods, we have shown that in CHO-K1 cells haponin interacts with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is one of the vital glycolytic enzymes with a diverse set of noncanonical functions.

Protein tyrosine kinase panel as a tool for anticancer drug design.

The discovery of the pharmaceutical potential of small molecule inhibitors of oncogenic protein tyrosine kinases is one of the directions in target therapy in oncology. Presently, investigations aiming at developing new therapeutically important inhibitors have to be based on a combination of computational and experimental approaches including biochitalicical, cell-based or in silico screening and the study of the three-dimensional structure of the kinase active center, in complex with an inhibitor, using crystallography and X-ray analysis or molecular modeling. This work is an example of a combination of inhibitor experimental search with the computational analysis of the potential mechanism of the inhibitors' action, which allowed to propose the 2-hydroxyphenol group as a scaffold for the design of new tyrosine kinase inhibitors.

Granulocyte differentiation inducer, hexapeptide HLDF-6, decreases cytotoxic effect of tumor necrosis factor on HL-60 cell line.

The effect of hexapeptide HLDF-6, the granulocytic differentiation inducer, on the tumor necrosis factor alpha (TNF-alpha)-induced differentiation and apoptosis of human promyelocytic leukemia HL-60 cells has been investigated. Costimulation of HL-60 cells with HLDF-6 and TNF-alpha enhanced granulocyte differentiation, whereas the level of monocyte differentiation remained unchanged; however, the cytotoxic action of TNF-alpha on these cells decreased. The protective effect of HLDF-6 peptide did not depend on activation of NF-kappaB (nuclear transcription factor). Since HLDF-6 peptide decreases the number of cells entering apoptosis caused by C(2)-ceramide, a mediator of TNF-induced apoptosis, and also reduces TNF-alpha-mediated activation of caspase-3, we have proposed the hypothesis that HLDF-6 increases resistance of HL-60 cells to the TNF-alpha cytotoxic effect due to inhibition of some stages of mitochondria-dependent apoptotic signaling.

[L-Glutamic acid modulates the cytotoxic effect of tumor necrosis factor in the HL-60 cell line].

L-Glutamic acid was shown to increase the stability of cells of the HL-60 line of human promyelocyte leukemia to the cytotoxic action of tumor necrosis factor alpha (TNF-alpha) due to the inhibition of apoptotic and NF-kappaB-activating cascades induced by this cytokine. At the same time, L-glutamic acid increases the TNF-alpha-mediated differentiating signal and the accompanying enhancement of the phosphatidylinositol-specific phospholipase C activity. Therefore, it is a promising agent for the reduction of total toxicity and inflammatory processes during treatment with TNF-alpha. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

[Surgical remodeling of the left ventricle in ischemic cardiomyopathy].

The paper covers early and long-term results of left ventricle geometric reconstruction and myocardial revascularization in 44 patients with ischemic cardiomyopathy. The authors have established clinical and hemodynamic criteria of ischemic cardiomyopathy, indications for surgical treatment and a surgical technique. The article presents a new approach to correction of mitral insufficiency in the given category of patients. According to the results of the study, the necessary conditions for a surgical intervention to be successful are: intraoperative monitoring of central hemodynamics using Swan-Ganz catheter, evaluation of myocardial contractile function by means of transoesophageal echocardiography and wide use of preoperative intraaortic balloon pumping. Geometric reconstruction results in normalization of left ventricle shape and its end-systolic and end-diastolic volumes, marked increase of ejection fraction and improvement of central hemodynamics. The stability of the long-term results has been confirmed in a 4-year follow-up.

[Precursors of HLDF differentiation factor and ribosomal protein RPS21 have a common N-terminal sequence]. / Predshestvennik faktora differentsirovki HLDP i ribosomnyi belok RPS21 imeiut obshchuiu N-kontsevuiu posledovatel'nost'.

The mature differentiation factor HLDF, isolated from culture fluid, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48-50 and 100-102, i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and the S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Cloning and characterization of the human ribosomal protein S21 gene [. / Klonirovanie i kharakteristika gena ribosomnogo belka S21 cheloveka [(letter)].

A full-size functional gene encoding the human ribosomal protein S21 was cloned and characterized. Its nucleotide sequence, exon-intron organization, and transcription initiation site were determined. The gene comprises 1417 bp and is composed of six exons and five introns. Like most known genes of mammalian ribosomal proteins, it lacks the canonical TATA- and CAAT sequences in the promoter region and harbors potential binding sites for transcription factors both upstream and downstream the transcription initiation site. The first intron of the rpS21 gene is located in the 5'-untranslated region. The transcription initiation site is at a 53-bp distance from the ATG codon, and the initiation cytidine is surrounded by polypyrimidine tracts. The 5'-flanking region contains two repeats belonging to the Alu-S and Alu-J families.

[Nucleotide sequence of cDNA and organization of the gene for alpha-subunit of photoreceptor phosphodiesterase of cyclic GMP in human retinal cones]. / Nukleotidnaia posledovatel'nost' kDNK i organizatsiia gena alpha'-sub''edinitsy fotoretseptornoi fosfodiésterazy tsiklicheskogo GMP kolbochek setchatki glaza cheloveka.

Five clones were isolated from a human retina cDNA library whose cDNA inserts allowed reconstruction of the total sequence of the human clone cGMP phosphodiesterase alpha'-subunit cDNA comprising 3455 bp. The protein's deduced sequence contains 858 amino acids residues with molecular mass 99,169 Da. A substantial homology was revealed between the amino acid sequence of the human cones cGMP-phosphodiesterase alpha'-subunit and the corresponding sequences of alpha, beta, and alpha' subunits of visual cGMP-phosphodiesterase of bovine, murine, chicken and human retinas. Four recombinant bacteriophages were isolated from a genomic library whose inserts made it possible to reconstruct a 32-kb fragment of the human cones cGMP-phosphodiesterase alpha'-subunit gene. 5'-Flanking region of the gene and first 14 exons, encoding an N-terminal segment of the protein, along with the adjacent intron segments were sequenced.

The human rod photoreceptor cGMP phosphodiesterase beta-subunit. Structural studies of its cDNA and gene.

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed alpha-, beta- and alpha'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4-22 of the PDE beta-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the beta-subunit gene was observed between exons 20 and 21 which led to the formation of the beta'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE beta'-subunit in human retina.

[Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]. / Strukturnye issledovaniia kDNK i gena beta-sub''edinitsy fosfodiésterazy cGMP iz cetchatki cheloveka.

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.