Involvement of a mitochondrial phosphatase in the regulation of ATP production and insulin secretion in pancreatic beta cells

Reversible phosphorylation is the cell's most prevalent form of posttranslational modification, yet its role in the regulation of mitochondrial functions is poorly understood. We have discovered that a member of the dual-specific protein tyrosine phosphatase (DS-PTP) family, PTPMT1 (PTP localized to the Mitochondrion 1) resides nearly exclusively in mitochondria. PTPMT1 is targeted to the mitochondrion by an N-terminal signal sequence and is found anchored to the matrix face of the inner membrane. Knockdown of PTPMT1 expression in the pancreatic insulinoma cell line INS-1 832/13 alters the mitochondrial phosphoprotein profile and markedly enhances both ATP production and insulin secretion. These data define PTPMT1 as a potential drug target for the treatment of type II diabetes and strengthen the notion that mitochondria are an underappreciated site of signaling by reversible phosphorylation.     

Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that?whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in?vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.     

Metabolic Regulation by the Mitochondrial Phosphatase PTPMT1 Is Required for Hematopoietic Stem Cell Differentiation

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Highlights? PTPMT1 depletion causes cell cycle delay and differentiation block in HSCs ? The HSC pool in PTPMT1 knockout mice is drastically (～40-fold) expanded ? Mitochondrial metabolism is altered and AMPK is highly activated in knockout HSCs ? PTPMT1 PIP substrates directly enhance fatty-acid-induced activation of UCP2     

The Pseudophosphatase MK-STYX Physically and Genetically Interacts with the Mitochondrial Phosphatase PTPMT1

We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.     

Identification of a Mammalian-type Phosphatidylglycerophosphate Phosphatase in the Eubacterium Rhodopirellula baltica

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.     

Firing up mitochondrial activities with PTPMT1

Pagliarini et al (2005) recently identified a new mitochondrial specific protein tyrosine phosphatase, PTPMT1. This report comments on its consequences for mitochondrial function and on its potential to act as a therapeutic target in diabetes and cancer.     

PTPMT1: connecting cardiolipin biosynthesis to mitochondrial function

Cardiolipin, a phospholipid component of the inner mitochondrial membrane, is required for mitochondrial metabolism. In this issue, Zhang et?al. (2011) highlight a critical role for PTPMT1, a mitochondrial phosphatase, in cardiolipin biogenesis and possibly in cardiolipin deficiency diseases. Their findings also unveil a yet uncharacterized pathway affecting cell growth.     

Phosphatidylglycerophosphate phosphatase is required for root growth in Arabidopsis

Phosphatidylglycerol (PG) is an indispensable lipid class in photosynthetic activity. However, the importance of PG biosynthesis in non-photosynthetic organs remains elusive. We previously identified phosphatidylglycerophosphate phosphatase 1 (PGPP1), which catalyzes the last step of PG biosynthesis in Arabidopsis thaliana. In the present report, we noted considerably shorter roots of the pgpp1-1 mutant compared to the wild type. We observed defective order of columella cells in the root apices, which was complemented by introducing the wild-type PGPP1 gene. Although PGPP1 is chloroplast-localized in leaf mesophyll cells, we observed mitochondrial localization of PGPP1 in root cells, suggesting possible dual targeting of PGPP1. Moreover, we identified previously uncharacterized 2 protein tyrosine phosphatase-like proteins as functional PGPPs. These proteins, designated PTPMT1 and PTPMT2, complemented growth and lipid phenotypes of Δgep4, a Saccharomyces cerevisiae mutant of PGPP. The ptpmt1-1 ptpmt2-1 exhibited no visible phenotype; however, the pgpp1-1 ptpmt1-1 ptpmt2-1 significantly enhanced the root phenotype of pgpp1-1 without further affecting the photosynthesis, suggesting that these newly found PGPPs are involved in the root phenotype. Radiolabeling experiment of mutant roots showed that decreased PG biosynthesis is associated with the mutation of PGPP1. These results suggest that PG biosynthesis is required for the root growth.     

Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Evaluating scoring functions for docking and designing beta-secretase inhibitors

Several simple scoring methods were examined for 2 series of beta-secretase (BACE-1) inhibitors to identify a docking/scoring protocol which could be used to design BACE-1 inhibitors in a drug discovery program. Both the PLP1 score and MMFFs interaction energy (E(inter)) performed as well or better than more computationally intensive methods for a set of substrate-based inhibitors, while the latter performed well for both sets of inhibitors.     

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.     

Potent mechanism-based inhibitors for matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.     

Design, synthesis and biological activity of sphingosine kinase 2 selective inhibitors

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell survival. SphK phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has been implicated in cancer growth and survival. SphK exists as two different isotypes, namely SphK1 and SphK2, which play different roles inside the cell. In this report, we describe SphK inhibitors based on the immunomodulatory drug, FTY720, which is phosphorylated by SphK2 to generate a S1P mimic. Structural modification of FTY720 provided a template for synthesizing new inhibitors. A diversity-oriented synthesis generated a library of SphK inhibitors with a novel scaffold and headgroup. We have discovered subtype selective inhibitors with K(i)'s in the low micromolar range. This is the first report describing quaternary ammonium salts as SphK inhibitors.     

Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput screening platform

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1−/−) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1−/− cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1−/− cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.