Biomarkers of extracellular matrix metabolism (MMP-9 and TIMP-1) and risk of stroke, myocardial infarction, and cause-specific mortality: cohort study

Objective

Turnover of the extracellular matrix in all solid organs is governed mainly by a balance between the degrading matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). An altered extracellular matrix metabolism has been implicated in a variety of diseases. We investigated relations of serum levels of MMP-9 and TIMP-1 to mortality risk from an etiological perspective.

Design

The prospective Uppsala Longitudinal Study of Adult Men (ULSAM) cohort, followed from 1991–1995 for up to 18.1 years. A random population-based sample of 1,082 71-year-old men, no loss to follow-up. Endpoints were all-cause (n = 628), cardiovascular (n = 230), non-cardiovascular (n = 398) and cancer mortality (n = 178), and fatal or non-fatal myocardial infarction (n = 138) or stroke (n = 163).

Results

Serum MMP-9 and TIMP-1 levels were associated with risk of all-cause mortality (Cox proportional hazard ratio [HR] per standard deviation 1.10, 95% confidence interval [CI] 1.03–1.19; and 1.11, 1.02–1.20; respectively). TIMP-1 levels were mainly related to risks of cardiovascular mortality and stroke (HR per standard deviation 1.22, 95% CI 1.09–1.37; and 1.18, 1.04–1.35; respectively). All relations except those of TIMP-1 to stroke risk were attenuated by adjustment for cardiovascular disease risk factors. Relations in a subsample without cardiovascular disease or cancer were similar to those in the total sample.

Conclusion

In this community-based cohort of elderly men, serum MMP-9 and TIMP-1 levels were related to mortality risk. An altered extracellular matrix metabolism may be involved in several detrimental pathways, and circulating MMP-9 or TIMP-1 levels may be relevant markers thereof.     

A novel dimer-tetramer transition captured by the crystal structure of the HIV-1 Nef

HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nef_core) domains respectively. Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nef_core in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nef_core dimers (open conformation) swing out with a relative displacement of ∼22 Å and rotation of ∼174° to form the ‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nef_core—Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.     

Synthesis, structure and biological evaluation of bis salicylaldehyde-4(N)-ethylthiosemicarbazone ruthenium(iii) triphenylphosphine

Bis salicylaldehyde-4(N)-ethylthiosemicarbazone ruthenium(iii) triphenylphosphine [Ru(Sal-etsc)(H-Sal-etsc)(PPh(3))] was synthesized and structurally characterized by spectral and X-ray crystallographic studies and it showed 100% inhibition on the DPPH radical. It also exhibited a significant lymphocyte activity and inhibitory effect on the lung carcinoma A549 cell.     

Characterization of the HslU chaperone affinity for HslV protease

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.     

Large-scale prediction of function shift in protein families with a focus on enzymatic function

Protein function shift can be predicted from sequence comparisons, either using positive selection signals or evolutionary rate estimation. None of the methods have been validated on large datasets, however. Here we investigate existing and novel methods for protein function shift prediction, and benchmark the accuracy against a large dataset of proteins with known enzymatic functions. Function change was predicted between subfamilies by identifying two kinds of sites in a multiple sequence alignment: Conservation-Shifting Sites (CSS), which are conserved in two subfamilies using two different amino acid types, and Rate-Shifting Sites (RSS), which have different evolutionary rates in two subfamilies. CSS were predicted by a new entropy-based method, and RSS using the Rate-Shift program. In principle, the more CSS and RSS between two subfamilies, the more likely a function shift between them. A test dataset was built by extracting subfamilies from Pfam with different EC numbers that belong to the same domain family. Subfamilies were generated automatically using a phylogenetic tree-based program, BETE. The dataset comprised 997 subfamily pairs with four or more members per subfamily. We observed a significant increase in CSS and RSS for subfamily comparisons with different EC numbers compared to cases with same EC numbers. The discrimination was better using RSS than CSS, and was more pronounced for larger families. Combining RSS and CSS by discriminant analysis improved classification accuracy to 71%. The method was applied to the Pfam database and the results are available at http://FunShift.cgb.ki.se. A closer examination of some superfamily comparisons showed that single EC numbers sometimes embody distinct functional classes. Hence, the measured accuracy of function shift is underestimated.     

NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.     

Retinoid metabolism during development of liver cirrhosis

The changes in retinoid metabolism have been documented in liver cirrhosis. However, the dynamic alterations in levels of this vitamin between circulation and liver during development of the liver cirrhosis are not well understood. The aim of this study was to measure retinoids in the liver and circulation in parallel, during and after development of cirrhosis induced by carbon tetrachloride and thioacetamide. Retinoid levels were measured by HPLC. A decrease in retinaldehyde and total retinol, together with an increase in retinoic acid was evident in liver from both carbon tetrachloride or thioacetamide treated rats within a month after initiation of treatment. Activity of enzymes involved in retinoid metabolism such as retinaldehyde oxidase, retinaldehyde dehydrogenase, and retinaldehyde reductase were decreased in the liver. In parallel, levels of retinol and retinaldehyde in the serum were increased while retinoic acid was decreased. This study indicates that during development of cirrhosis, there is reciprocal transfer of retinoid metabolites between the circulation and the liver.     

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.     

Linoleic acid-induced endothelial activation: role of calcium and peroxynitrite signaling

Hypertriglyceridemia, an important risk factor of atherosclerosis, is associated with increased circulating free fatty acids. Research to date indicates that linoleic acid (LA), the major fatty acid in the American diet, may be atherogenic by activating vascular endothelial cells. However, the exact signaling mechanisms involved in LA-mediated proinflammatory events in endothelial cells still remain unclear. We previously reported increased superoxide formation after LA exposure in endothelial cells. The objective of the present investigation is to determine the role of calcium and peroxynitrite in mediating the proinflammatory effect of LA in vascular endothelial cells. LA exposure increased intracellular calcium, nitric oxide, and tetrahydrodiopterin levels as well as the expression of E-selectin. Inhibiting calcium signaling using 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and heparin decreased the expression of E-selectin. Also, LA-mediated nuclear factor kappa B activation and E-selectin gene expression were suppressed by Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (a superoxide scavenger), N(G)-monomethyl-l-arginine (an endothelial nitric oxide synthase inhibitor), and 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride (a peroxynitrite scavenger). LA exposure resulted in increased nitrotyrosine levels, as observed by Western blotting and immunofluorescence. Our data suggest that the proinflammatory effects of LA can be mediated through calcium and peroxynitrite signaling.