Exploring inhibitor binding at the S' subsites of cathepsin L.

We report a series of noncovalent, reversible inhibitors of cathepsin L that have been designed to explore additional binding interactions with the S' subsites. The design was based on our previously reported crystal structure that suggested the possibility of engineering increased interactions with the S' subsites ( Chowdhury et al. J. Med. Chem. 2002, 45, 5321-5329 ). A representative of these new inhibitors has been co-crystallized with mature cathepsin L, and the structure has been solved and refined at 2.2 A. The inhibitors described in this work extend farther into the S' subsites of cathepsins than any inhibitors reported in the literature thus far. These interactions appear to make use of a S3' subsite that can potentially be exploited for enhanced specificity and/or affinity.

Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.

Carboxy-monopeptidase substrate specificity of human cathepsin X.

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.

Up-regulation of cathepsin X in prostate cancer and prostatic intraepithelial neoplasia.

BACKGROUND: Evidence is accumulating that several proteases are involved in prostate cancer progression. A locus which is often amplified in prostate cancer is the chromosomal region 20q13. Interestingly, one of the genes encoding the cysteine protease cathepsin X maps to this region. The aim of this study was to assess the expression pattern of cathepsin X in malignant and non-malignant prostatic tissue samples. METHODS: Matched malignant and non-malignant tissue specimens were obtained from 56 men after radical prostatectomy. Cathepsin X was quantified at both protein and mRNA levels using several detection methods: Western blotting, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. Furthermore, genomic DNA was analyzed by PCR for possible gene amplification. RESULTS: Immunohistochemical analysis of formalin-fixed, paraffin-embedded sections of radical prostatectomy specimens was performed utilizing a polyclonal antibody against human procathepsin X and revealed staining of acinar basal cells in normal prostate glands. Prostatic intraepithelial neoplasias (PINs) and prostate carcinomas stained highly positive for cathepsin X, showing a significant difference to the staining of normal prostate glands. In contrast, relatively weak and heterogeneous staining was observed for cathepsins F, B, and L. Up-regulation of cathepsin X at the protein level was confirmed by Western blotting. No statistically significant difference was observed at the mRNA level. PCR of genomic DNA revealed that cathepsin X up-regulation most likely occurs in the absence of genomic amplification. CONCLUSIONS: The high expression levels of cathepsin X both in PIN and invasive adenocarcinomas of the prostate suggest that cathepsin X may play a role in the early tumorigenesis of prostate cancer. Further studies are needed to define the utility of this cysteine protease as a diagnostic marker for the early detection of prostate cancer.

Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity.

Influence of group A streptococcal acid glycoprotein on expression of major virulence factors and internalization by epithelial cells.

A single transposon insertion upstream to the open-reading-frame identified as the streptococcal acid glycoprotein (sagp) gene rendered a Tn916 isolate of Streptococcus pyogenes with elevated susceptibility to internalization by the epithelial cells. The role of SAGP in S. pyogenes internalization was further studied using isogenic mutant containing an in-frame deletion within the sagp gene. The sagp mutant displayed slower growth-rate and showed 5-fold higher internalization efficiency than the parent strain. Transcription of sagp at the logarithmic phase, but not at the stationary phase of the growth was repressed by csrR, the global regulator gene. At the same time, mutation of the sagp gene partially decreased the transcription of hasA, a gene that is required for capsule synthesis. The mutation had no effect on transcription of the emm3 gene, encoding for the M protein. The most striking effect of the sagp mutation was a down-regulation of the streptococcal pyrogenic exotoxin B (SpeB) at both translational and transcriptional level. Treatment of the SAGP mutant cells with the exogenous mSpeB (mature protease) only partially reduced their susceptibility to internalization. The exogenous mSpeB was more effective in reducing the internalization efficiency of a speB mutant and brought it to the level observed for the parent strain. In overall, results show that CsrR, directly or indirectly, affects the expression of SAGP, and that the SAGP modulates expression of not only SpeB, but also other genes that facilitate S. pyogenes internalization.