Realizing the allosteric potential of the tetrameric protein kinase A RIα holoenzyme

PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the?D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RIα that contains most of the N-Linker, RIα(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RIα mutation localized to a novel interface unique to the tetramer.     

Phase behaviour of transkarbam 12

Transkarbam 12 (T12), the carbamic acid salt of omega-aminocaproic acid dodecyl ester, is a recently synthesized substance, whose high permeation enhancing activity through the human skin was found for certain drugs. In this work, the thermotropic phase behaviour of T12 has been studied by means of various techniques, namely, DSC, FTIR and FT-Raman spectroscopy, X-ray powder diffraction, and DRS. The temperature development of the X-ray reflections as well as of the conformationally sensitive Raman bands and the IR bands have been observed. At room temperature, the hydrocarbon chains of T12 exhibit a highly ordered structure, arranged in an orthorhombic perpendicular subcell. On heating, two transitions occur at 54 and at 66 degrees C. The first transition is related to the disruption of the carbamate structure and changes in the polar head group. The other transition represents the melting of hydrocarbon chains and the subsequent release of carbon dioxide. The time required for the rebinding of carbon dioxide and the reformation of the carbamate structure is dependent upon numerous factors and it was not possible to precisely determine the length of this process.     

Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.     

Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling

Organs and organelles represent core biological systems in mammals, but the diversity in protein composition remains unclear. Here, we combine subcellular fractionation with exhaustive tandem mass spectrometry-based shotgun sequencing to examine the protein content of four major organellar compartments (cytosol, membranes [microsomes], mitochondria, and nuclei) in six organs (brain, heart, kidney, liver, lung, and placenta) of the laboratory mouse, Mus musculus. Using rigorous statistical filtering and machine-learning methods, the subcellular localization of 3274 of the 4768 proteins identified was determined with high confidence, including 1503 previously uncharacterized factors, while tissue selectivity was evaluated by comparison to previously reported mRNA expression patterns. This molecular compendium, fully accessible via a searchable web-browser interface, serves as a reliable reference of the expressed tissue and organelle proteomes of a leading model mammal.     

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM₂) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380–450 nm.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.     

Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>: purification,characterization, and application to increase purine nucleoside production

Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-α-d-ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC 2.7.6.1). Class I PRSs are widespread in bacteria and mammals, are highly conserved among different organisms, and are negatively regulated by two end products of purine biosynthesis, adenosine 5′-diphosphate (ADP) and guanosine 5′-diphosphate (GDP). The D52H, N114S, and L129I mutations in the human PRS isozyme I (PRS1) have been reported to cause uric acid overproduction and gout due to allosteric deregulation and enzyme superactivity. In this study, to find feedback-resistant Bacillus amyloliquefaciens PRS, the influence of the D58H, N120S, and L135I mutations (corresponding to the D52H, N114S, and L129I mutations in PRS1, respectively) on PRS enzymatic properties has been studied. Recombinant histidine-tagged wild-type PRS and three mutant PRSs were expressed in Escherichia coli, purified, and characterized. The N120S and L135I mutations were found to release the enzyme from ADP and GDP inhibition and significantly increase its sensitivity to inorganic phosphate (P_i) activation. In contrast, PRS with the D58H mutation exhibited nearly identical sensitivity to ADP and GDP as the wild-type protein and had a notably greater P_i requirement for activation. The N120S and L135I mutations improved B. amyloliquefaciens and Bacillus subtilis purine nucleoside-producing strains.     

Computer-aided design of polyketides with the required properties

An approach to rational design of new polyketides with the required spectrum of biological activity has been proposed. We have developed the BioGenPharm software, which generates combinatorial libraries of polyketides based on the user-defined input parameters, performs prediction of biological activity spectra for the generated structures and selection of molecuels with the required properties. PASS algorithm has been applied for prediction of polyketide activity spectra (http://www.ibmc.msk.ru/PASS). Validation of PASS prediction ability for polyketides was performed vs. the evaluation set containing 242 natural macrolides from the Dictionary of Natural Products. The mean prediction accuracy was 75.5%. The problem of choice of the cutting points for probability of the presence of activity (Pa), which provides optimal combination of such parameters as sensitivity, specificity, concordance has been considered. Applicability of the described method has been illustrated by generation of a virtual library of the erythromycin analogues and selection of substances with low probability of the hepatotoxic effect.