Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

Identification of amino acid residues of the matrix metalloproteinase-2 essential for its selective inhibition by beta-amyloid precursor protein-derived inhibitor

The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.     

Critical temperatures for the interaction of free fatty acids with the erythrocyte membrane

Non-esterified long-chain fatty acids reduce the extent of hypotonic hemolysis at a certain low concentration range but cause hemolysis at higher concentrations. This biphasic behavior was investigated at different temperatures (0-37 degrees C) for lauric (12:0), myristic (14:0), palmitoleic (16:1), oleic (cis-18:1) and elaidic (trans-18:1) acids. The results are summarized as follows: (A) the fatty acids examined exhibit a high degree of specificity in their thermotropic behavior; (B) oleic acid protects against hypotonic hemolysis even at the highest concentrations, up to 15 degrees C, when it becomes hemolytic, but only in a limited concentration range; (C) elaidic acid does not affect the osmotic stability of erythrocytes up to 20 degrees C, when it starts protecting: above 30 degrees C, it becomes hemolytic at the highest concentrations; (D) palmitoleic acid is an excellent protecting agent at all temperatures in a certain concentration range, becoming hemolytic at higher concentrations; (E) lauric acid protects up to 30 degrees C and becomes hemolytic only above this temperature; (F) myristic acid exhibits an extremely unusual behavior at 30 and 37 degrees C by having alternating concentration ranges of protecting and hemolytic effects; (G) there is a common critical temperature for hemolysis at 30 degrees C for saturated and trans-unsaturated fatty acids; (H) the initial slope of Arrhenius plots of percent hemolysis at the concentration of maximum protection is negative for cis-unsaturated fatty acids and positive for saturated and trans-unsaturated fatty acids.     

In silico scaffold evaluation and solid phase approach to identify new gelatinase inhibitors

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Effects of Fatty Acids,Lipase Activator,Phospholipids and Related Substances on the Lipase Production by Candida paralipolytica

Unsaturated long-chain fatty acids such as oleic, linoleic and ricinoleic acid except for elaidic acid were effective on the lipase production by Candida paralipolytica, but saturated fatty acids were not effective. When elaidic and myristic acids were dissolved in n-hexadecane to be dispersed in a liquid state, they became to be effective on the lipase production. These results suggest that long-chain fatty acids of a liquid state are effective on the lipase production. Lipase activator A and phosphatidylethanolamine stimulated the effect of fatty acids on the lipase production. Effects of sterols and surface-active substances on the lipase production were also reported.

A weak tributyrin-hydrolyzing activity, in the absence of fatty acids, was found in the yeast cell.     

Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568-631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity.     

Modulation of gelatinase activity correlates with the dedifferentiation profile of regenerating salamander limbs.

Remodeling of extracellular matrix (ECM) is one of the key events in many developmental processes. In the present study, a temporal profile of gelatinase activities in regenerating salamander limbs was examined zymographically. In addition, the effect of retinoic acid (RA) on these enzyme activities was examined to relate the pattern-duplicating effect of RA in limb regenerates with gelatinase activities. During regeneration, various types of gelatinase activities were detected, and these activities were at their maximum levels at the dedifferentiation stage. Upon treatment with chelating agents EDTA and 1,10-phenanthroline, the enzyme activities were inhibited indicating that those enzymes are likely matrix metalloproteinases (MMPs). Considering the molecular sizes and the decrease of molecular sizes by treatment with p-aminophenylmercuric acetate, an artificial activator of proMMP, some of the gelatinases expressed during limb regeneration are presumed to be MMP-2 and MMP-9. In RA-treated regenerates, overall gelatinase activities increased, especially the MMP-2-like gelatinase activity which increased markedly. These results suggest that MMP-2-like and MMP-9-like gelatinases play a role in ECM remodeling during regeneration, and that gelatinases are involved in the excessive dedifferentiation after RA treatment.     

The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema.

Large chondroitinsulphate-containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP-9 and proMMP-2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP-2 and MMP-9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.     

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants.

The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.     

Effects of long-chain fatty acids on the inhibition by antimycin of respiration in hepatocytes and isolated mitochondria from rat liver

A previous study [Berry, M. N., Gregory, R. B., Grivell, A. R. & Wallace, P. G. (1983) Eur. J. Biochem. 131, 215-222] suggested that long-chain fatty acid (palmitate) oxidation by hepatocytes was less sensitive than short-chain fatty acid (hexanoate) oxidation to inhibition by a given concentration of antimycin. Re-examination of this phenomenon showed that palmitate oxidation by hepatocytes could be depressed by antimycin to the same degree as other NAD+-linked substrates, only if the concentration of the inhibitor was raised 2-4-fold. The presence of palmitate also reduced the sensitivity to antimycin of hepatocytes metabolizing lactate or pyruvate. Over the range of fatty acids tested, butyrate (C4) to stearate (C18), only long-chain (greater than C10) fatty acids endowed cells with decreased sensitivity towards antimycin. 2-Bromopalmitate, a non-metabolizable fatty acid, and inhibitor of fatty acid oxidation, also decreased the inhibitory effect of antimycin in cells, suggesting that long-chain fatty acids per se rather than their metabolites, reverse the inhibition by antimycin. Moreover, another inhibitor of fatty acid oxidation, 2-tetradecylglycidic acid, did not diminish the effects of palmitate. Succinate oxidation in isolated mitochondria that had been inhibited by a low concentration of antimycin could be restored by subsequent addition of palmitate or other long-chain fatty acids such as dodecanoate, tetradecanoate and oleate under conditions where fatty acid oxidation was prevented. 2-Bromopalmitate, likewise partially restored antimycin-depressed succinate oxidation. This amelioration of antimycin inhibition was counteracted by the addition of more antimycin and was not seen upon addition of defatted bovine serum albumin, palmitoylcarnitine or octanoate. The total amount of antimycin bound to mitochondria was not affected by the presence of palmitate. The data suggest that long-chain fatty acids are able to interact with the mitochondrial inner membrane in a manner which can relieve the inhibitory effect of antimycin, whether the antimycin is added to the cell or mitochondrial suspension before or after fatty acid addition.     

Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes.

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.     

Requirement for omega and (omega;-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega;-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega;-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega;-1)/omega ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega;-1)-hydroxylation inside the access channel of active site.     

The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.     

Simultaneous presence of unsaturation and long alkyl chain at P'1 of Ilomastat confers selectivity for gelatinase A (MMP-2) over gelatinase B (MMP-9) inhibition as shown by molecular modelling studies

Structural analogues of Ilomastat (Galardin), containing unsaturation(s) and chain extension carrying bulky phenyl group or alkyl moieties at P'1 were synthesized and purified by centrifugal partition chromatography. They were analyzed for their inhibitory capacity towards MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14, main endopeptidases involved in tumour progression. Presence of unsaturation(s) decreased the inhibitory potency of compounds but, in turn increased their selectivity for gelatinases. 2b and 2d derivatives with a phenyl group inhibited preferentially MMP-9 with IC50 equal to 45 and 38 nM, respectively, but also display activity against MMP-2 (IC50 equal to 280 and 120 nM, respectively). Molecular docking computations confirmed affinity of these substances for both gelatinases. With aims to obtain a specific gelatinase A (MMP-2) inhibitor, P'1 of Ilomastat was modified to carry one unsaturation coupled to an alkyl chain with pentylidene group. Docking studies indicated that MMP-2, but not MMP-9, could accommodate such substitution; indeed 2a proved to inhibit MMP-2 (IC50=123 nM), while displaying no inhibitory capacity towards MMP-9.     

Heterocycle-based MMP inhibitors with P2' substituents

Potent and selective inhibition of matrix metalloproteinases was demonstrated for a series of sulfonamide-based hydroxamic acids. The design of the heterocyclic sulfonamides incorporates a six- or seven-member central ring with a P2' substituent that can be modified. Binding interactions of this substituent at the S2' site are believed to contribute to high inhibitory potency against stromelysin, collagenase-3 and gelatinases A and B, and to provide selectivity against collagenase-1 and matrilysin. An X-ray structure of a stromelysin inhibitor complex was obtained to provide insights into the SAR and selectivity trends observed for the series.     

Effects of fatty acid chain length and saturation on gastric inhibitory polypeptide release in obese hyperglycaemic (ob/ob) mice

Plasma gastric inhibitory polypeptide (GIP) responses to equimolar intragastrically administered emulsions of fatty acids (2.62 mmol/7.5 ml/kg) were examined in 18 h fasted obese hyperglycaemic (ob/ob) mice. Propionic acid (C3:0), a saturated short-chain fatty acid, and capric acid (C10:0), a saturated medium chain fatty acid, did not signilicantly stimulate GIP release. However, the saturated long-chain fatty acid stearic acid (C18:0), and especially the unsaturated long-chain fatty acids oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids produced a marked GIP response. The results show that chain length and to a lesser extent the degree of saturation are important determinants of fatty acid-stimulated GIP release. The GIP-release action of long-chain, but not short-chain, fatty acids may be r e l a t e d to differences in their intracellular handling.     

Structure- and configuration-dependent effects of C18 unsaturated fatty acids on the chicken and sheep erythrocyte membrane

High concentrations of unsaturated fatty acids are known to cause hemolysis. At low concentrations, however, unsaturated cis fatty acids have been found to protect erythrocytes against hypotonic hemolysis. In the present experiments we examined the effect of oleic (18:1), linoleic (18:2), linolenic (18:3), and elaidic (18:1) acid on the osmotic fragility of chicken and sheep erythrocytes, which markedly differ in their resistance to osmotic rupture. The results are summarized as follows: (A) The phenomenon of stabilization was observed in both species alike. (B) Interaction of cells with the fatty acids under isotonic conditions led to a persistent stabilization, i.e., the cells remained more resistant against osmolysis even after several washings. (C) Oleic and elaidic acid protected against osmotic rupture with a high degree of specificity. Linoleic and linolenic acid were much less protective. Thus, this effect appears to be specific for one double bond. (D) Contrary to the unsaturated fatty acids with cis configuration, elaidic acid with the trans configuration showed no biphasic behaviour, and even at the highest concentrations applied no hemolysis was observed.     

The role of gelatinases in colorectal cancer progression and metastasis

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully.     

Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria

The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.