The biochemical and morphological changes in the myocardium of patients with an acute surgical vascular pathology (based on data from early autopsies)]

Biochemical and morphological disorders of glucose 6-phosphate dehydrogenase (G-6-PDH) were studied in the myocardium of 9 patients who had died from different vascular surgical diseases. The inhibition of G-6-PDH activity most prominent in lung artery thromboembolism was shown biochemically. Histological and histoenzymological findings demonstrate low G-6-PDH activity in different myocardial regions and solitary defects of cardiomyocytes. The data obtained evidence significant sensibility of the myocardium in surgical patients to different influences.     

ING2 recruits histone methyltransferase activity with methylation site specificity distinct from histone H3 lysines 4 and 9

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.     

Interactions of fluorinated surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.     

More compact protein globules exhibit slower folding rates

We have demonstrated that, among proteins of the same size, alpha/beta proteins have on the average a greater number of contacts per residue due to their more compact (more "spherical") structure, rather than due to tighter packing. We have examined the relationship between the average number of contacts per residue and folding rates in globular proteins according to general protein structural class (all-alpha, all-beta, alpha/beta, alpha+beta). Our analysis demonstrates that alpha/beta proteins have both the greatest number of contacts and the slowest folding rates in comparison to proteins from the other structural classes. Because alpha/beta proteins are also known to be the oldest proteins, it can be suggested that proteins have evolved to pack more quickly and into looser structures.     

Stereoselectivity of binding of alpha-(N-benzylamino)benzylphosphonic acids to prostatic acid phosphatase

The inhibition effects of enantiomerically pure alpha-(N-benzylamino)benzylphosphonic acids and their derivatives on human prostatic acid phosphatase have been investigated. As expected, (R)-alpha-(N-benzylamino)benzylphosphonic acid demonstrated higher affinity for the enzyme than (S)-enantiomer. At the same time, (1R,2S)-phenyl[(1-phenylethyl)amino]methylphosphonic acid was found to be a significantly weaker inhibitor than its (1S,2R)-analogue. The enantioselectivity has been explained using a molecular modeling approach by computational docking of inhibitors into active center of prostatic acid phosphatase.     

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay

Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.     

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.     

Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.     

Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate

Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser(10). In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.     

Structural insight into the interaction between platelet integrin alphaIIbbeta3 and cytoskeletal protein skelemin

Skelemin is a large cytoskeletal protein critical for cell morphology. Previous studies have suggested that its two-tandem immunoglobulin C2-like repeats (SkIgC4 and SkIgC5) are involved in binding to integrin beta3 cytoplasmic tail (CT), providing a mechanism for skelemin to regulate integrin-mediated signaling and cell spreading. Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin alphaIIbbeta3. Here, we show that skelemin IgC45 domains form a complex not only with integrin beta3 CT but also, surprisingly, with the integrin alphaIIb CT. Chemical shift mapping experiments demonstrate that both membrane-proximal regions of alphaIIb and beta3 CTs are involved in binding to skelemin. NMR structural determinations, combined with homology modeling, revealed that SkIgC4 and SkIgC5 both exhibited a conserved Ig-fold and both repeats were required for effective binding to and attenuation of alphaIIbbeta3 cytoplasmic complex. These data provide the first molecular insight into how skelemin may interact with integrins and regulate integrin-mediated signaling and cell spreading.