Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.     

Interleukin-18: biological properties and clinical implications

IL-18, originally identified as interferon-gamma inducing factor (IGIF), is related to the IL-1 family in terms of its structure, processing, receptor, signal transduction pathway and pro-inflammatory properties. IL-18 is also functionally related to IL-12, as it induces the production of Th1 cytokines and participates in cell-mediated immune cytotoxicity. This review summarizes the recent advances in the understanding of IL-18 structure, processing, receptor expression and immunoregulatory functions, and focuses on the role of IL-18 modulation in tumours, infections, and autoimmune and inflammatory diseases.     

The probability distribution for a random match between an experimental-theoretical spectral pair in tandem mass spectrometry

Proteomic techniques are fast becoming the main method for qualitative and quantitative determination of the protein content in biological systems. Despite notable advances, efficient and accurate analysis of high throughput proteomic data generated by mass spectrometers remains one of the major stumbling blocks in the protein identification problem. We present a model for the number of random matches between an experimental MS-MS spectrum and a theoretical spectrum of a peptide. The shape of the probability distribution is a function of the experimental accuracy, the number of peaks in the experimental spectrum, the length of the interval over which the peaks are distributed, and the number of theoretical spectral peaks in this interval. Based on this probability distribution, a goodness-of-fit tool can be used to yield fast and accurate scoring schemes for peptide identification through database search. In this paper, we describe one possible implementation of such a method and compare the performance of the resulting scoring function with that of SEQUEST. In terms of speed, our algorithm is roughly two orders of magnitude faster than the SEQUEST program, and its accuracy of peptide identification compares favorably to that of SEQUEST. Moreover, our algorithm does not use information related to the intensities of the peaks.     

Mutational and structural analyses of the hinge region of membrane type 1-matrix metalloproteinase and enzyme processing

Membrane type 1 (MT1)-matrix metalloproteinase (MMP) is a major mediator of collagen degradation in the pericellular space in both physiological and pathological conditions. Previous evidence has shown that on the cell surface, active MT1-MMP undergoes autocatalytic processing to a major membrane-tethered 44-kDa product lacking the catalytic domain and displaying Gly285 at its N terminus, which is at the beginning of the hinge domain. However, the importance of this site and the hinge region in MT1-MMP processing is unknown. In the current study, we generated mutations and deletions in the hinge of MT1-MMP and followed their effect on processing. These studies established Gly284-Gly285 as the main cleavage site involved in the formation of the 44-kDa species. However, alterations at this site did not prevent processing. Instead, they forced downstream cleavages within the stretch of residues flanked by Gln296 and Ser304 in the hinge region, as determined by the processing profile of various hinge deletion mutants. Also, replacement of the hinge of MT1-MMP with the longer MT3-MMP hinge did not prevent processing of MT1-MMP. Molecular dynamic studies using a computational model of MT1-MMP revealed that the hinge region is a highly motile element that undergoes significant motion in the highly exposed loop formed by Pro295-Arg302 consistent with being a prime target for proteolysis, in agreement with the mutational data. These studies suggest that the hinge of MT1-MMP evolved to facilitate processing, a promiscuous but compulsory event in the destiny of MT1-MMP, which may play a key role in the control of pericellular proteolysis.     

High-potency human immunodeficiency virus vaccination leads to delayed and reduced CD8+ T-cell expansion but improved virus control

CD8(+) T lymphocytes are thought to play an important role in the control of acute and chronic human immunodeficiency virus infections. However, there is a significant delay between infection and the first observed increase in virus-specific CD8(+) T-cell numbers. Prior to this time, viral kinetics are not significantly different between controls and vaccinees. Surprisingly, higher initial virus-specific CD8(+) T-cell numbers lead to a longer delay prior to initial CD8(+) T-cell expansion, and slower CD8(+) T-cell increases. Nevertheless, higher initial CD8(+) T-cell numbers were associated with reduced peak and chronic viral loads and reduced CD4(+) T-cell depletion.     

Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines

Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.     

Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases

We have investigated the molecular basis of selective and complete C1s deficiency in 2-year-old girl with complex autoimmune diseases including lupus-like syndrome, Hashimoto's thyroiditis, and autoimmune hepatitis. This patient's complement profile was characterized by the absence of CH50 activity, C1 functional activity <10%, and undetectable levels of C1s Ag associated with normal levels of C1r and C1q Ags. Exon-specific amplification of genomic DNA by PCR followed by direct sequence analysis revealed a homozygous nonsense mutation in the C1s gene exon XII at codon 534, caused by a nucleotide substitution from C (CGA for arginine) to T (TGA for stop codon). Both parents were heterozygous for this mutation. We used the new restriction site for endonuclease Fok-1 created by the mutation to detect this mutation in the genomic DNA of seven healthy family members. Four additional heterozygotes for the mutation were identified in two generations. Our data characterize for the first time the genetic defect of a selective and complete C1s deficiency in a Caucasian patient.     

Two tightly linked QTLs modify tomato sugar content via different physiological pathways

Congenic lines that differ in a single defined chromosome segment are useful for the study of complex phenotypes, as they allow isolation of the effect of a particular quantitative trait locus (QTL) from those of the entire genome. We conducted high-resolution QTL mapping of a 9-cM introgression, originating from the wild tomato species Lycopersicon pennellii, in two extremely different genetic and physiological backgrounds. In the "indeterminate" glasshouse background we identified only a single QTL ( Brix9-2-5) that affects the total soluble solids of the fruit [mainly sugars, measured in Brix units (B)]. This QTL was previously delimited within the gene for an apoplastic invertase, Lin5, that modulates sugar partitioning to the fruit. Analysis of the effects of the same chromosome segment in "determinate", open-field tomatoes, revealed two QTLs, 0.3 cM apart: the fruit-specific Brix9-2-5 that affects B only, and the shoot-specific PW9-2-5, which accounts for an altered growth habit resulting in increases in plant weight, yield, and B. This study highlights the power of the congenic approach for dissecting developmental pathways leading to complex phenotypes.     

MMP-2 colocalizes with caveolae on the surface of endothelial cells

We examined the spatial distribution of MMP-2 on the surface of human endothelial cells using immunofluorescence and confocal microscopy. Staining endothelial cells with MMP-2-specific antibodies revealed a punctate labeling at the basolateral side of the cell periphery, which colocalized with patches of caveolin-1, a major constituent of the caveolae. This colocalization was confirmed by immunogold electron microscopy. MT1-MMP, TIMP-2, and the alphavbeta3 integrin exhibited a similar pattern of staining, with pericellular patches that colocalized with either MMP-2 or caveolin-1. The presence of MT1-MMP and TIMP-2 in caveolae patches could be seen only after treatment with concanavalin A, which induced MMP-2 activation but had no noticeable effect on the pattern or intensity of MMP-2 immunostaining. In contrast, MMP-9 and TIMP-1 staining showed a pattern completely different from that of MMP-2 and TIMP-2, with positive spots uniformly distributed throughout the cell body. Our data show that MMP-2, its activator the MT1-MMP, and its proposed receptor, the alphavbeta3 integrin, are all targeted to the same membrane microdomains on the endothelial cell, thereby restricting matrix proteolysis to a limited microenvironment at the cell surface.