Reactive site-modified tissue inhibitor of metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A.
(1/182)
Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the alpha-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form. (+info)
Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed.
(2/182)
Matrix metalloproteinases (MMPs) catalyze extracellular matrix degradation. Control of their activity is a promising target for therapy of diseases characterized by abnormal connective tissue turnover. MMPs are expressed as latent proenzymes that are activated by proteolytic cleavage that triggers a conformational change in the propeptide (cysteine switch). The structure of proMMP-2 reveals how the propeptide shields the catalytic cleft and that the cysteine switch may operate through cleavage of loops essential for propeptide stability. (+info)
Is 100KF an isoform of hemopexin? Immunochemical characterization of the vasoactive plasma factor 100KF.
(3/182)
The human vasoactive plasma factor 100KF has been proposed to play a role in minimal change disease in relapse. Since preliminary data suggested similarity between 100KF and the human plasma glycoprotein hemopexin (Hx), this study was conducted to compare 100KF with purified Hx for sequence homology, immunostaining properties in Western and dot-blot assays, ability to affect glomerular ecto-ATPase and glomerular polyanions in vitro, as well as their glomerular permeability increasing effect following alternate perfusion into the rat kidney ex vivo. 100KF was purified from normal pooled plasma according to standard chromatographic techniques, and from the same batch Hx was prepared using affinity chromatography. A second batch of Hx was prepared directly from human serum according to a standard protocol. (For comparison, additional Hx samples obtained from other centers were also included in the study.) The results show: (1) 100% homology of 100KF with plasma Hx after internal sequence analysis; (2) positive staining of the eluate with both monoclonal and polyclonal anti-Hx IgG as well as anti-100KF IgG in dot-blot assays, and similar bands on Western blotting using the same antibodies; (3) affection of glomerular polyanions and glomerular ecto-ATPase after incubation of kidney tissue with either 100KF or Hx (1.5 respectively 1.0 mg/ml; 1.0 h, 37 degrees C), as detected by computerized histochemical quantification; and (4) significant enhancement of urinary protein leakage after Hx perfusion followed by diluted rat serum into the rat kidney ex vivo (Hx: 210.65+/-49.79 microg protein leakage per min versus heat-inactivated Hx control: 112.2+/-49.18 microg per min [both n = 6]). From these data and from the observation that both Hx and 100KF activity can be inhibited by serine protease inhibitors but not by broad spectrum collagenase inhibitors, it is concluded that Hx may be closely related or identical to the active moiety of 100KF. (+info)
Defective recovery and severe renal damage after acute hemolysis in hemopexin-deficient mice.
(4/182)
Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes. (+info)
Induction of experimental proteinuria in vivo following infusion of human plasma hemopexin.
(5/182)
BACKGROUND: The human plasma constituent hemopexin (Hx), following incubation with renal tissue, is able to induce glomerular alterations in vitro that are similar to those seen in minimal change disease (MCD). Whether this acute phase reactant is also able to induce proteinuria and minimal change-like alterations in vivo is questioned. METHODS: In the first set of experiments, Hx (4.0 mg in 5.0 mL saline) or equal amounts of control fraction, that is, heat-inactivated Hx (HI-Hx), were infused into conscious rats (N = 6) that had been surgically equipped with a cannula inserted into the suprarenal artery (SRA), enabling direct contact of the infusate and the renal microvasculature. Each animal received HI-Hx at day 1 for 15 minutes (flow rate 20.0 mL/h), subsequently followed by saline for seven hours (Flow rate 5.0 mL/h), after which the cannula was disconnected. At day 2, identical infusions in the same rat were carried out, using native Hx. Urine samples collected every 30 minutes during the experiments were monitored for protein content using standard methods. In the second set of experiments, unilateral perfusion was done ex vivo in anesthetized rats with Hx (N = 5) or HI-Hx (N = 3; 1.5 mg/mL; 4.0 mL during 6 min). After reconnection of the circulation, urine samples of both kidneys were collected every 30 minutes during five hours via ureter cannulation. Urinary protein (expressed as the difference in excretion between perfused and nonperfused kidney) was calculated in mg/24 h. In additional experiments, rats were sacrificed two hours after perfusion of Hx or heat-inactivated (control) Hx (first set of experiments) or after five hours (second set of experiments), and kidneys were processed for immunohistochemical and ultrastructural examination. RESULTS: The results of experiment 1 show a significant increase of proteinuria after Hx infusion versus HI-Hx (means +/- SD, 41.91 +/- 16.01 mg/24 h vs. control, 21.22 +/- 5.69 mg/24 h; P +info)
Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3.
(6/182)
Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response. (+info)
A noncanonical WD-repeat protein from the cyanobacterium Synechocystis PCC6803: structural and functional study.
(7/182)
SYNECHOCYSTIS: PCC6803 possesses several open reading frames encoding putative WD-repeat proteins. One, the Hat protein, is involved in the control of a high-affinity transport system for inorganic carbon that is active when the cells are grown under a limiting concentration of this carbon substrate. The protein is composed of two major domains separated by a hydrophobic linker region of 20 amino acid residues. The N-terminal domain of Hat has no homolog in standard databases and does not display any particular structural features. Eleven WD repeats have been identified in the C-terminal moiety. The region encompassing the four terminal WD repeats is essential for growth under a limiting inorganic carbon regime. The region encompassing the two most terminal WD repeats is required for the activity of the high-affinity transport system. However, because the Hat protein is located in the thylakoids, it should not be itself an element of the transport system. The structural organization of the WD-containing domain of Hat was modeled from the crystal structure of the G protein beta subunit (with seven WD repeats) and of hemopexin (a structural analog with four blades). Functional and structural data argue in favor of an organization of the Hat WD moiety in two subdomains of seven and four WD repeats. The C-terminal 4-mer subdomain might interact with another, yet unknown, protein/peptide. This interaction could be essential in modulating the stability of the 4-mer structure and, thus, the accessibility of this subdomain, or at least of the region encompassing the last two WD repeats. (+info)
Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain.
(8/182)
In view of the essential role of the hemopexin domain of the traditional interstitial collagenases, MMP-1, -8, -13 and MT1-MMP (MMP-14), in determining specific collagen cleavage we have studied the function of this domain in MMP-2, relative to that of the fibronectin-like domain that promotes gelatinolysis. Although the fibronectin-like domain promotes avid binding to collagen, our data demonstrate that the catalytic and hemopexin domains of MMP-2 are sufficient to effect the critical step in cleavage of rat type I collagen into 3/4 and 1/4 fragments. The mechanism of MMP-2 cleavage of collagen proceeds in two phases, the first resembling that of the interstitial collagenases, followed by gelatinolysis, promoted by the fibronectin-like domain. (+info)