S28 peptidases: lessons from a seemingly 'dysfunctional' family of two

<p>Abstract</p> <p>A recent paper in <it>BMC Structural Biology </it>reports the crystal structure of human prolylcarboxypeptidase (PRCP), one of the two members of the S28 peptidase family. Comparison of the substrate-binding site of PRCP with that of its family partner, dipeptidyl dipeptidase 7 (DPP7), helps to explain the different enzymatic activities of these structurally similar proteins, and also reveals a novel apparent charge-relay system in PRCP involving the active-site catalytic histidine.</p> <p>See research article: <url>http://www.biomedcentral.com/1472-6807/10/16/</url></p> <p>Commentary</p> <p>The S28 serine peptidase family is something of an enzymatic odd couple. While showing low sequence similarity to all proteins except each other, the two known family members appear to be at odds functionally; one, prolylcarboxypeptidase (PRCP), is a carboxypeptidase that cleaves single hydrophobic residues from the carboxyl termini of proteins that end with a Pro-X motif (where X is any hydrophobic amino acid), while the other, human dipeptidyl peptidase (DPP7), is an aminopeptidase that cleaves amino-terminal X-Pro dipeptides. The structural basis of this orthogonal specificity would undoubtedly be interesting, and a recent report in <it>BMC Structural Biology </it>from the Merck Global Structural Biology group (Soisson <it>et al</it>. <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>) has now met that expectation. In addition they reveal a new wrinkle to the iconic catalytic triad common to most serine hydrolases.</p> <p>The practical pharmaceutical interest in both these enzymes as potential drug targets is at present speculative. PRCP can inactivate a number of peptide hormones, such as angiotensin II, III and prekallikrein, implicating a role for the enzyme in hypertension, tissue proliferation and smooth-muscle growth. These properties suggest that this enzyme may well be a useful target for hypertension and anti-inflammatory therapy <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. Another (non-S28 family) dipeptidyl dipeptidase (DPP4) is a major drug target in type 2 diabetes, and Merck has already developed a successful inhibitor of DPP4, the anti-hyperglycemic drug sitagliptin, for the treatment of type 2 diabetes. The DPP enzymes are rich in biological functions and other drug targets emerging from the group are possible <abbrgrp><abbr bid="B3">3</abbr></abbrgrp>.</p

Phosphorylation of SOS1 on tyrosine 1196 promotes its RAC GEF activity and contributes to BCR-ABL leukemogenesis

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1fl/flmice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL

Discovery That Theonellasterol a Marine Sponge Sterol Is a Highly Selective FXR Antagonist That Protects against Liver Injury in Cholestasis

Background: The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions. Principal Findings: Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTa, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4. Conclusions: FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis

Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?

The laboratory mouse has been widely used to test the efficacy of schistosome vaccines and a long list of candidates has emerged from this work, many of them abundant internal proteins. These antigens do not have an additive effect when co-administered, or delivered as SWAP homogenate, a quarter of which comprises multiple candidates; the observed protection has an apparent ceiling of 40–50 %. We contend that the low level of maturation of penetrating cercariae (~32 % for Schistosoma mansoni) is a major limitation of the model since 68/100 parasites fail to mature in naïve mice due to natural causes. The pulmonary capillary bed is the obstacle encountered by schistosomula en route to the portal system. The fragility of pulmonary capillaries and their susceptibility to a cytokine-induced vascular leak syndrome have been documented. During lung transit schistosomula burst into the alveolar spaces, and possess only a limited capacity to re-enter tissues. The acquired immunity elicited by the radiation attenuated (RA) cercarial vaccine relies on a pulmonary inflammatory response, involving cytokines such as IFNγ and TNFα, to deflect additional parasites into the alveoli. A principal difference between antigen vaccine protocols and the RA vaccine is the short interval between the last antigen boost and cercarial challenge of mice (often two weeks). Thus, after antigen vaccination, challenge parasites will reach the lungs when both activated T cells and cytokine levels are maximal in the circulation. We propose that “protection” in this situation is the result of physiological effects on the pulmonary blood vessels, increasing the proportion of parasites that enter the alveoli. This hypothesis will explain why internal antigens, which are unlikely to interact with the immune response in a living schistosomulum, plus a variety of heterologous proteins, can reduce the level of maturation in a non-antigen-specific way. These proteins are “successful” precisely because they have not been selected for immunological silence. The same arguments apply to vaccine experiments with S. japonicum in the mouse model; this schistosome species seems a more robust parasite, even harder to eliminate by acquired immune responses. We propose a number of ways in which our conclusions may be tested

Identification of an allosteric binding site for RORγt inhibition

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors