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.PRL (for phosphatase of regenerating liver) phosphatases constitute a novel class of small tyrosine phosphatases involved in the modulation of cell growth. Initial studies identified PRL-1 as an intermediate-early gene expressed in the early response of regenerating liver tissue to mitogens (1). Overexpression of this protein was shown to lead to cellular transformation (1-3). The biological role of PRL-1 is tissue-dependent. Its overexpression is associated with cell proliferation in the liver (1) but with differentiation of epithelial cells in the digestive system (4). The closely related phosphatases PRL-2 and PRL-3 are also involved in growth regulation, proliferation, and cell invasion (3, 5, 6). All three proteins are prenylated at their C terminus, which critically affects their cellular localization and function (6 -8). As shown for the human PRL-2, the role of PRLs is associated with the regulation of progression through mitosis, and their cellular localization is likely controlled by the cell cycle (8).PRL phosphatases are widely distributed in eukaryotes. In humans, PRL-1 and PRL-2 are ubiquitously expressed in various tissues (6), whereas PRL-3 is normally expressed in cardiac and skeletal muscles (5). Comprising typically only 140 -180 amino acids, PRLs are among the smallest phosphatases. They consist of a single catalytic domain lacking any auxiliary docking/regulatory domains other than the prenylation site at the C terminus. PRLs contain the protein-tyrosine phosphatase (PTPase) 1 active consensus motif HCXXGXXR, referred to as the P-loop; however, their primary sequence shows only remote similarity to phosphatases in other regions. Tyrosine-specific phosphatases a...
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.
The structures of Candida rugosa lipase-inhibitor complexes demonstrate that the scissile fatty acyl chain is bound in a narrow, hydrophobic tunnel which is unique among lipases studied to date. Modeling of triglyceride binding suggests that the bound lipid must adopt a "tuning fork" conformation. The complexes, analogs of tetrahedral intermediates of the acylation and deacylation steps of the reaction pathway, localize the components of the oxyanion hole and define the stereochemistry of ester hydrolysis. Comparison with other lipases suggests that the positioning of the scissile fatty acyl chain and ester bond and the stereochemistry of hydrolysis are the same in all lipases which share the alpha/beta-hydrolase fold.
The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have k cat /K M values ranging from 1200 to 7700´m 21´s21 , making them very good endopeptidase substrates for SCP.
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.
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