A major feature of the structure of alpha 1-antitrypsin is a five-stranded A-sheet into which the reactive center loop inserts after cleavage. We describe here the effect of the Z mutation (342Glu to Lys) at the head of the fifth strand of the A-sheet on the mobility of the reactive center loop and hence on the physical properties of the antitrypsin molecule. The mutant Z but not the normal M antitrypsin spontaneously polymerizes at 37 degrees C by a mechanism involving the insertion of the reactive center loop of one molecule into the A-sheet of a second. It is demonstrated that Z antitrypsin polymerized after incubation with 1.0 M guanidinium chloride at 37 degrees C at the same rate as M antitrypsin. Reducing the temperature to 4 degrees C favored the formation of the L-state in M antitrypsin in which the loop is stably incorporated into the A-sheet, but resulted in loop-sheet polymerization in Z antitrypsin. Z, like M antitrypsin, undergoes the S to R transition, but we show that the accompanying change in thermal stability results from loop-sheet polymerization (S) which can be prevented by the insertion of the cleaved strand of the reactive center loop into the A-sheet (R). Z antitrypsin has a reduced association rate constant with neutrophil elastase [(5.3 +/- 0.06) x 10(7) and (1.2 +/- 0.02) x 10(7) M-1 s-1 for M and Z, respectively], but both M and Z antitrypsin had Ki values of less than 5 pM.(ABSTRACT TRUNCATED AT 250 WORDS)

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Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-mer peptide (homologous to PI4-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to PI4-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with the SIC sequence of antithrombin or by an unrelated peptide representing residues 26-3 1 of cholecystokinin. The s IC or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361 + Cys, Ser 283 + Cys, was engineered for the purpose of forming a disulfide bond between SIC and s2C to prevent movement of SIC. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed' linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Abstract.Apart from SERPINB2 and SERPINB5, the roles of the remaining 13 members of the human SERPINB family in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of each SERPINB gene effectively suppressed the invasiveness and motility of malignant cancer cells. Among all of the genes, the SERPINB1, SERPINB5 and SERPINB7 genes were more potent, and the inhibitory effect was further enhanced by co-expression of any two of them. In addition, single treatment of the synthetic peptides corresponding to the P5-P5' sequences of the reactive center loop (RCL) of SERPINB1, SERPINB5 or SERPINB7 markedly suppressed the invasive and migratory properties of the cancer cells in a dose-dependent manner. More significantly, combination treatment of these peptides in cancer cells further improved the suppressive effect by 20-40%. Here, we determined the expression of all SERPINB family members in lung and breast cancer patients, and identified those members with potent inhibitory ability toward invasion and migration, and designed RCL-derived peptides to suppress the malignancy of cancer cells. Forced re-expression of these anti-invasive SERPINB genes or application of the SERPINB RCL-peptides may provide a reasonable strategy against lethal cancer metastasis. IntroductionCancer metastasis is the leading cause of morbidity and mortality in cancer patients. It is a highly complex process, including cell detachment, migration, invasion, circulation in blood vessels, adhesion, colonization at other sites and formation of secondary tumors (1). Prior to tumor cell detachment from the primary site, which leads to cell migration and invasion in the metastasis process, the extracellular matrix (ECM) microenvironment must be degraded by proteases, such as urokinase plasminogen activator (uPA), uPA receptor (uPAR) and the plasmin network (2,3) and matrix metalloproteinases (MMPs) (4). On the other hand, protease inhibitors negatively regulate the proteolysis process in cancer metastasis, e.g. plasminogen activator inhibitors (PAIs), PAI-1 (SERPINE1) and PAI-2 (SERPINB2) against uPA/uPAR/plasmin network and the tissue inhibitor of matrix metalloproteinases (TIMPs), TIMP-1 to TIMP-4 against MMPs.Serine protease inhibitors (serpins) regulate many physiological processes, such as blood coagulation, fibrinolysis, inflammation, complement activation and cell migration (5). Based on their phylogenic relationships, the superfamily is divided into 16 different clades (A-P), in which human serpins are the first 9 clades (A-I) (6). The clade B serpins (SERPINB family) is the largest one within the human serpin superfamily. It contains 13 genes located on chromosome 6p25 (SERPINB1, SERPINB6 and SERPINB9) and 18q21 (the remaining members of the family). Unlike circulating serpins, the SERPINB family genes lack the N and C terminus extension regions common to other serpin...

The human kallikrein 8 (KLK8) gene, a member of the human tissue kallikrein gene family, encodes a serine protease. The KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. We found that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppresses their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA reduced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and staining of cells for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with the detection of circulating tumor cells by quantitative PCR for the human Alu sequence showed that KLK8 suppresses tumor growth and invasion in vivo. Finally, studies of clinical specimens from patients with non-small cell lung cancer showed that the time to postoperative recurrence was longer for early-stage patients (stages I and II) with high KLK8 expression (mean, 49.9 months) than for patients with low KLK8 expression (mean, 22.9 months). Collectively, these findings show that KLK8 expression confers a favorable clinical outcome in nonsmall cell lung cancer by suppressing tumor cell invasiveness.