Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Cleavage of the 45-kDa gelatin-binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40-kDa FN-type-1V collagenase A degrades heat-denatured type-I collagen, laminin and also native collagen type IV. The 27-kDa FN-type-IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N-terminal sequence VYQPQPH-(residues 262 -268 of fibronectin) but, contrary to the FN-type-IV collagenase A, the FN-type;IV collagenase B has lost the C-terminal region of type I repeats, where the major gelatin-binding determinants of fibronectin are located. The FN-type-IV collagenases A and B are sequentially similar to the middle domain (domain 11) of collagenase type IV, secreted by H-ras-transformed human bronchial epithelial cells.Substrate and inhibition specificity of FN-type-IV collagenase A and B are different from those of FNgelatinase and FN-laminase, isolated previously from the central and C-terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix-degrading metalloproteinases.Fibronectin is an adhesion protein that is present in both extracellular matrix and blood plasma. This large 540-kDa glycoprotein is composed by two nearly identical polypeptide chains, each comprised of multiple specific sites for binding various ligands including collagens, fibrin and heparin. Each polypeptide chain of fibronectin is composed of three types of sequential repeats (reviewed in [l -31).The collagen-binding domain is located near the N-terminus within a 45-kDa region that contains four type-I and two type-I1 repeats. Whereas the type-I repeats are found also in other functional domains of fibronectin, the type-I1 units are specific for the gelatin-binding domain [4-61. It was therefore suggested that these type-I1 repeats are responsible for the interaction between fibroncctin and collagen(s), a mechanism which is fundamental to the organization of extracellular matrix.Recent studies, however, have provided evidence that the collagen-binding site is located between the second type-I1 unit and the adjacent type-I unit, within a 12-kDa fragment including the sequence -AAHE- [7], which is also found in the metal-binding site of matrix-degrading metalloproteinases. Moreover, it was recently shown that the major gelatin-binding determinants of fibronectin are located within a 21-kDa region of two type-I repeats that occurs in the C-terminal extremity of this domain 181. The initial idea for the present study was to search for the mechanism of interaction between fibronectin and its ligand, collagen. In particular, we have examined the question of whether this interaction might be analogous to the interaction of a zymogen with its potential substrate. We have previously demonstrated a similar mechanism of interaction between trypsin...

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2' into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262 -265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond KS5-DS6 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca2+-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.Fibronectin is the best known cell adhesion protein that can also be incorporated into the extracellular matrix. It occurs at high level in plasma and other body fluids. Fibronectin contains several structural domains, each of which exhibits various biological functions. The fibronectin molecule (550 kDa) is composed of two almost identical polypeptide chains linked by two disulfide bonds near the COOH-terminus. Each chain bears a number of structural repeats named type I, 11, and 111. The type I and 11 repeats are characterized by a high content of disulfide bonds that are absent in the type I11 repeats (Fig. 1).The N-terminal 29-kDa domain of fibronectin is composed by five type I repeats which are organized into a finger structure. It bears one of the fibrin-binding sites, one of the heparin-binding sites of fibronectin and one of the sites for transglutaminase-activated crosslinking. The N-terminal domain binds Staphylococcus aureus, DNA and the C-terminal heparin-binding domain of adjacent fibronectin molecules to form polymers (reviewed in [l -31).In our previous work we described the isolation and characterization of two matrix-degrading proteinases, FNgelatinase and FN-laminase. The enzymes were localized in the central and C-terminal domains, respectively 14-71. In this paper we describe the generation of a new proteolytic activity during the degradation process of the N-terminal fibronectin fragment.