X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.     

Assessment of the Relationship between Self-Declared Ethnicity,Mitochondrial Haplogroups and Genomic Ancestry in Brazilian Individuals

In populations that have a high degree of admixture, such as in Brazil, the sole use of ethnicity self-declaration information is not a good method for classifying individuals regarding their ethnicity. Here, we evaluate the relationship of self-declared ethnicities with genomic ancestry and mitochondrial haplogroups in 492 individuals from southeastern Brazil. Mitochondrial haplogroups were obtained by analyzing the hypervariable regions of the mitochondrial DNA (mtDNA), and the genomic ancestry was obtained using 48 autosomal insertion-deletion ancestry informative markers (AIM). Of the 492 individuals, 74.6% self-declared as White, 13.8% as Brown and 10.4% as Black. Classification of the mtDNA haplogroups showed that 46.3% had African mtDNA, and the genomic ancestry analysis showed that the main contribution was European (57.4%). When we looked at the distribution of mtDNA and genomic ancestry according to the self-declared ethnicities from 367 individuals who self-declared as White, 37.6% showed African mtDNA, and they had a high contribution of European genomic ancestry (63.3%) but also a significant contribution of African ancestry (22.2%). Of the 68 individuals who self-declared as Brown, 25% showed Amerindian mtDNA and similar contribution of European and African genomic ancestries. Of the 51 subjects who self-declared as black, 80.4% had African mtDNA, and the main contribution of genomic ancestry was African (55.6%), but they also had a significant proportion of European ancestry (32.1%). The Brazilian population had a uniform degree of Amerindian genomic ancestry, and it was only with the use of genetic markers (autosomal or mitochondrial) that we were able to capture Amerindian ancestry information. Additionally, it was possible to observe a high degree of heterogeneity in the ancestry for both types of genetic markers, which shows the high genetic admixture that is present in the Brazilian population. We suggest that in epidemiological studies, the use of these methods could provide complementary information.     

Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products

A large proportion of the genes in any plant genome encode enzymes of primary and specialized (secondary) metabolism. Not all plant primary metabolites, those that are found in all or most species, have been identified. Moreover, only a small portion of the estimated hundreds of thousand specialized metabolites, those found only in restricted lineages, have been studied in any species. The correlative analysis of extensive metabolic profiling and gene expression profiling has proven a powerful approach for the identification of candidate genes and enzymes, particularly those in secondary metabolism. The final characterization of substrates, enzymatic activities, and products requires biochemical analysis, which has been most successful when candidate proteins have homology to other enzymes of known function. The challenges are to identify new types of enzymes and to develop biochemical techniques that are suitable for large-scale analysis.     

Effects of non-thermal plasma on mammalian cells

Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of intracellular reactive oxygen species (ROS). We have utilized γ-H2AX to detect DNA damage induced by non-thermal plasma and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks; however, plasma does not lead to formation of bulky adducts/thymine dimers.     

Corticoadrenal activity regulates in rat betaine-homocysteine <Emphasis Type="Italic">S</Emphasis>-methyltransferase expression with opposites effects in liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras

A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.