Showing papers by "Gary A. Silverman published in 1998"

Cross-Class Inhibition of the Cysteine Proteinases Cathepsins K, L, and S by the Serpin Squamous Cell Carcinoma Antigen 1: A Kinetic Analysis†

Abstract: The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

Abstract: The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Scca1 and scca2 are proteinase inhibitors that map to the serpin cluster at 18q21.3

Abstract: The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and approximately 10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).

Delineation of the breakpoint at 18q21.1 in a cell line (karpas1106) derived from mediastinal b-cell lymphoma by fluorescence in situ hybridization with multiple YAC clones

Abstract: The breakpoint of the 18q21 translocation of B-cellnon-Hodgkin's lymphoma (NHL) cell line Karpas1106P was delineated by fluorescence in situ hybridization (FISH). Karpas1106P was derived from mediastinal lymphoblastic B-cell lymphoma and exhibited the immunophenotype characteristic of marginal-zone B-cell lymphoma (MZL): smIg+, pan-B antigen+, CD5−, CD10− and CD23−. The original G-banded karyotype showed a complex translocation containing t(X;18;13)(q28;q21;q12.1). Double-color FISH (DCFISH) with whole-chromosome-painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast artificial chromosome (YAC) clones defined t(X;18;13) as ider(X)t(X;18;13)(q28;q12.3q21.1;q12.1). The immunoglobulin-heavy-chain (IgH) gene was not involved in the chromosomal translocation as detected by DCFISH with VH and Cγ probes. By using contiguous YAC clones mapped from 18q12.3 to q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1. In Karpas1106P, the distal part of chromosome 18 from the breakpoint (18q21.1-qter) was deleted, showing loss of heterozygosity of this region. In addition, the chromosomal segment 18q21.1 was duplicated and inserted to ider(X)t(X;18;13) between Xq28 and 13q12.1 with maintaining its original orientation. The DNA sequence of the breakpoint region contained in y852H2 can serve as a candidate locus for further molecular dissection to identify the causative gene of MZL. Int. J. Cancer 78:100–105, 1998.© 1998 Wiley-Liss, Inc.

Preventive therapy for chronic lung disease.

Abstract: To the Editor We read with great interest the article by Stiskal and colleagues titled “α1-Proteinase Inhibitor Therapy for the Prevention of Chronic Lung Disease of Prematurity: A Randomized, Controlled Trial”1 As pointed out in the article, chronic lung disease of prematurity (CLD) or unresolved neonatal acute lung injury is a multifactorial disease, with lung immaturity, barovolutrauma, and oxygen toxicity being the most widely recognized and studied precedent factors CLD continues to be one of the most commonly encountered complications of prematurity despite surfactant displacement and improved ventilatory management strategies aimed at minimizing injury to the immature lung Even though evidence from numerous descriptive studies over the past 2 decades, using tracheal aspirate samples as the representative milieu of the airways, has suggested a role for inflammation and unfavorable proteinase/antiproteinase balance in the development of CLD, there has been considerable lag time in hypothesis testing in both the laboratory and clinical settings We, therefore, congratulate Stiskal and his colleagues on their effort to taking the proteinase/antiproteinase imbalance theory a step forward, to be …