A human fibroblast cDNA expression library was screened for cDNA clones giving rise to f lat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.Mutations of ras protooncogenes are found in a large variety of human tumors (1). It has been well established that Ras proteins are essential components in various intracellular signaling pathways involved in regulating gene expression and several other aspects of cellular behavior (2). Therefore, it is now important to find targets for these signals relevant to the expression of the malignant phenotype to understand the mechanism of cell transformation and to develop means to cure or prevent cancers.To this end, we have been isolating and characterizing genes that induce flat morphology (or ''flat reversion'') when expressed in a v-Ki-ras-transformed NIH 3T3 cell line, DT (3). The Krev-1 gene (4), also known as rap1A, which encodes a Ras-related protein containing a region identical to the effector domain of Ras, was isolated in a previous study (5) by using a plasmid-based human fibroblast cDNA expression library. Using a similar approach, Cutler et al. (6) isolated another transformation suppressor gene, rsp-1, encoding a leucinerich-repeat protein. Recently, we performed a similar screen of a human fibroblast cDNA expression library constructed with a new phagemid shuttle vector and obtained two cDNA clones exhibiting significant biological activities. One of these, clone CT124, was found to encode a truncated form of the MSX-2 homeobox protein, which induces flat reversion through a dominant-negative mechanism over the endogenous MSX-2 protein (7).Here we describe some properties of the other reversioninducing gene named RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) and its product. This reversioninducing gene is unique in that it encodes an extracellular protein with protease inhibitor-like domains and its expression is suppressed strongly in many tumors and cells transformed by various kinds of oncogenes. Restored expression of the RECK gene inhibits the invasive and metastatic activities of tumor cells. We also found that RECK negatively regulates matrix metalloproteinase-9 (MMP-9) (8) in two ways: suppression of MMP-9 secretion from the cells and direct inhibition of its enzymatic activity. T...

Mammalian transglutaminase (TGase) catalyzes covalent crosslinking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPD(P), QxP, and QxxDP; Factor XIIIa, QxxxWP (where x and represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.Transglutaminase (TGase, 2 2.3.2.13) is an enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and also attaches other primary amines to peptide-bound glutamines (1-5). To date, eight human TGase isozymes (Factor XIII, TGases 1-7) have been identified, comprising a large protein family with unique tissue distributions and physiological roles. Among the isozymes, TGase 2 and Factor XIII are two major members, although their locations and regulation differ. TGase 2 is expressed ubiquitously and has been implicated in many biological processes, including apoptosis, stabilization of the extracellular matrix, and regulation of growth factors (6, 7). Plasma Factor XIII is synthesized as a zymogen that comprises two A and two B subunits (8). The A subunit contains the catalytic domain and is converted by thrombin-dependent proteolysis into its active form during clot formation. Activated Factor XIII (Factor XIIIa) is involved in fibrin stabilization and wound healing (9).Generally, TGase reactions involve a Ca 2ϩ -dependent acyl transfer via a double displacement mechanism. In the first step, a glutamine ␥-carboxyamide group in the substrate binds to a cysteine at the active site, resulting in formation of a ␥-glutamylthioester bond and release of ammonia. Formation of the covalent acyl enzyme intermediate is the rate-limiting step. The -amino group of a peptide-bound lysine, as a nucleophilic substrate, binds to the acyl enzyme intermediate and then attacks the thioester bond, thereby generating an intermolecular isopeptide -(␥-glutamyl)lysine cross-link. Primary amines can replace lysine in transamidation reactions and become incorp...

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.

Peflin, a newly identified 30-kDa Ca 2؉ -binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We pre-

Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that K i values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.The cellular activity of a protease is the result of many regulatory mechanisms such as the concentration and compartmentalization of substrates, the enzyme itself, and its cognate inhibitors. Cystatins are the natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteases and have shown important regulatory and protective functions in cells and tissues against proteolysis by cysteine proteases of host, bacterial, and viral origin (1-3). The inhibitory activity of cystatins is regulated by a reversible, tight-binding interaction between the protease inhibitor and its target protease (4). Disturbance of the normal balance between cysteine proteases and their inhibitors at a wrong time and location can lead to several pathological conditions such as chronic inflammatory reactions (5), tumor malignancy (6), and faulty differentiation processes in the epidermis and hair follicle (7). Little is known on the specific biological functions of cystatin family members. However, mutations in the genes encoding the cystatin family members cystatin B and C cause neurological phenotypes in humans (8, 9).Cystatin M/E is a 14-kDa secreted protein that shares only 35% homology with other human type 2 cystatins. Nevertheless, it has a similar overall structure including the two characteristic intrachain disulfide bridges (10, 11). Expression of cystatin M/E is found to be restricted to ...