Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.     

Charcot-Marie-Tooth type 4B demyelinating neuropathy: deciphering the role of MTMR phosphatases

Charcot-Marie-Tooth type 4B (CMT4B) is a severe autosomal recessive neuropathy with demyelination and myelin outfoldings of the nerve. This disorder is genetically heterogeneous, but thus far, mutations in myotubularin-related 2 (MTMR2) and MTMR13 genes have been shown to underlie CMT4B1 and CMT4B2, respectively. MTMR2 and MTMR13 belong to a family of ubiquitously expressed proteins sharing homology with protein tyrosine phosphatases (PTPs). The MTMR family, which has 14 members in humans, comprises catalytically active proteins, such as MTMR2, and catalytically inactive proteins, such as MTMR13. Despite their homology with PTPs, catalytically active MTMR phosphatases dephosphorylate both PtdIns3P and PtdIns(3,5)P2 phosphoinositides. Thus, MTMR2 and MTMR13 may regulate vesicular trafficking in Schwann cells. Loss of these proteins could lead to uncontrolled folding of myelin and, ultimately, to CMT4B. In this review, we discuss recent findings on this interesting protein family with the main focus on MTMR2 and MTMR13 and their involvement in the biology of Schwann cell and CMT4B neuropathies.     

Identification and localization of a new human myotubularin-related protein gene, mtmr8, on 8p22-p23

Myotubularin and myotubularin-related proteins are dual-specificity phosphatases.Several myotubularin-related proteins have been identified in humans and mice. The members of the myotubularin protein family are highly conserved, from humans to yeast. Mutations in the human myotubularin gene (MTM1) lead to X-linked myotubular myopathy. Here we isolate and localize a novel putative myotubularin-related protein gene (MTMR8) on chromosome 8p22--p23,between the markers D8S550 and D8S265, by exon-trapping experiments and RT-PCR. Genomic sequencing revealed that the gene consists of 10 exons and spans approximately 43 kb. The corresponding cDNA is 7081 bp. The open reading frame predicts a protein of 549 amino acids and a calculated molecular mass of 63 kDa. Like myotubularin-related protein-5, MTMR8 has no dual-specificity phosphatase domain. It contains a double-helical motif similar to the SET interaction domain, which is thought to have a role in the control of cell proliferation.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.     

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation

The MTM (myotubularin)/MTMR (myotubularin-related) protein family is comprised of 15 lipid phosphatases, of which nine members are catalytically active. MTMs are known to play a fundamental role in human physiology as gene mutations can give rise to X-linked myotubular myopathy or Charcot-Marie-Tooth disease, which manifest in skeletal muscle or in peripheral neurons respectively. Interestingly, studies have shown MTMR2 and MTMR5, two MTM family members, to be highly expressed in the testis, particularly in Sertoli and germ cells, and knockout of either gene resulted in spermatogenic defects. Other studies have shown that MTMR2 functions in endocytosis and membrane trafficking. In the testis, MTMR2 interacts and co-localizes with c-Src/phospho-Src-(Tyr?1?), a non-receptor protein tyrosine kinase that regulates the phosphorylation state of proteins at the apical ES (ectoplasmic specialization), a unique type of cell junction found between Sertoli cells and elongating/elongated spermatids. In the present review, we highlight recent findings that have made a significant impact on our understanding of this protein family in normal cell function and in disease, with the emphasis on the role of MTMs and MTMRs in spermatogenesis. We also describe a working model to explain how MTMR2 interacts with other proteins such as c-Src, dynamin 2, EPS8 (growth factor receptor pathway substrate 8) and ARP2/3 (actin-related protein 2/3) at the apical ES and the apical TBC (tubulobulbar complex; tubular-like invaginations that function in the disassembly of the apical ES and in the recycling of its components) to regulate spermiation at late stage VIII of the seminiferous epithelial cycle.     

Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases

Phosphoinositides control many different processes required for normal cellular function. Myotubularins are a family of Phosphatidylinositol 3-phosphate (PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in patients suffering from X-linked myotubular myopathy and the MTMR2 gene in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease type 4B. MTM1 is a phosphatidylinositol phosphatase with reported specificity toward PtdIns3P, while the related proteins MTMR2 and MTMR3 hydrolyze both PtdIns3P and PtdIns(3,5)P2. We have investigated MTM1 and MTMR6 and find that they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric ring that is 12.5 nm in diameter, and it is a specific allosteric activator of MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 falling within a putative pleckstrin homology domain reduces the ability of the enzyme to respond to PtdIns5P. We propose that the myotubularin family of enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P functions in a positive feedback loop controlling their activity. These findings highlight the importance of regulated phosphatase activity for the control of phosphoinositide metabolism.     

Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.     

PTEN and myotubularin phosphatases: from 3-phosphoinositide dephosphorylation to disease

The phosphatase and tensin homolog deleted on chromosome ten (PTEN) and myotubularin (MTM1) represent subfamilies of protein tyrosine phosphatases whose principal physiological substrates are D3-phosphorylated inositol phospholipids. As lipid phosphatases, PTEN- and MTM1-related (MTMR) proteins dephosphorylate the products of phosphoinositide 3-kinases and antagonize downstream effectors that utilize 3-phosphoinositides as ligands for protein targeting domains or allosteric activation. Here, we describe the cellular mechanisms of PTEN and MTMR function and their role in the etiology of cancer and other human diseases.     

The structure and regulation of myotubularin phosphatases

The human neuromuscular diseases X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B are caused by mutations in myotubularin family proteins. The myotubularins are a unique subfamily of protein tyrosine phosphatases that utilize inositol phospholipids, rather than phosphoproteins, as substrates. Recent structural studies, including the first crystal structure of a myotubularin family protein, have defined the structural features that are characteristic of the family and revealed the molecular basis of their unique substrate specificity. Interestingly, the myotubularin family contains a subgroup of proteins that are catalytically inactive. Recent biochemical studies have established that the inactive myotubularins function as adaptors for the active members and play an important regulatory role within the family.     

FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain

Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently.     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Myotubularin regulates the function of the late endosome through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P(2) binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P(2).     

MTMR9 Increases MTMR6 Enzyme Activity, Stability, and Role in Apoptosis

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual specificity protein tyrosine phosphatase active site CX₅R motif (1–3). Eight members of the myotubularin family possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate (PtdIns-3-P)⁴ and phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P₂) at the D-3 position, and six members are not catalytically active because they lack the conserved cysteine residue in the protein tyrosine phosphatase motif that is required for activity. Interest in this group of proteins originated from the genetic evidence linking myotubularin, the founding member of this family, to myotubular myopathy, an X-linked disorder characterized by severe hypotonia and generalized muscle weakness (4). Subsequently, mutations in MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative disorder (5, 6).Despite near identical substrate specificity, biochemical and genetic evidence supports the hypothesis that myotubularin proteins are not redundant and have unique functions within cells (2, 7–9). The mechanisms by which loss of function of myotubularin proteins produce diseases are not known. Current evidence supports the hypothesis that each myotubularin protein regulates a specific pool of PtdIns-3-P and/or PtdIns-3,5-P₂, which in turn regulates a variety of cellular functions. Differences in tissue expression and subcellular localization play a role in the specificity of different myotubularins (10–15).The functions of myotubularin proteins are altered by the formation of heteromers between catalytically active and inactive members of the family. The initial biochemical purification of MTM1 demonstrated the presence of MTM1 homodimers and MTM1-3-phosphatase adapter protein (3PAP) heteromers (16), which was later described as MTMR12 (15, 17). MTMR2 was found to form heteromers with MTMR5 (13) and MTMR13 (18), and MTMR7 formed heteromers with MTMR9 (19). In each case, a catalytically active myotubularin protein interacted with an inactive protein. Heteromerization generated two important effects: increased catalytic activity of the active component (13, 15, 19, 20) and targeting of the heteromer to specific subcellular locations (15). Mutations in the inactive member MTMR13 result in a similar phenotype in patients as the mutations in its catalytically active binding partner MTMR2, indicating an indispensable role for the catalytically inactive subunit (21).Myotubularin proteins can be grouped into subfamilies based on homology. Closely related MTMR6, MTMR7, and MTMR8 comprise such a subfamily. We have previously characterized the interaction between mouse MTMR7 and MTMR9 proteins (19). In this report, we characterize the interaction between human MTMR6 and MTMR9. MTMR6 and MTMR9 have been shown to form a heteromeric complex in mouse and Caenorhabditis elegans (19, 22). MTMR6 has been shown to inhibit the activity of a calcium-activated potassium channel (type KCa3.1) (23, 24). Two screening experiments implicate MTMR6 as a regulator of apoptosis. By RNA microarray analysis, increased MTMR6 expression was observed in B cell chronic lymphoid leukemia cells with increased resistance to irradiation-induced apoptosis (25), whereas in an RNA interference screen in HeLa cells, decreased MTMR6 expression promoted apoptosis (26).Here we show that MTMR6 interacts with MTMR9 in vitro and in human cells. This interaction increases the phospholipid binding and enzymatic activity of MTMR6 in vitro. Co-expression of either subunit in cells dramatically increased the protein levels of the individual binding partners, suggesting that heteromer formation increases the stability of the proteins. Finally, MTMR9 was found to potentiate the effects of MTMR6 on apoptosis.     

Myotubularin phosphoinositide phosphatases, protein phosphatases, and kinases: their roles in junction dynamics and spermatogenesis

Spermatogenesis in the seminiferous epithelium of the mammalian testis is a dynamic cellular event. It involves extensive restructuring at the Sertoli-germ cell interface, permitting germ cells to traverse the epithelium from basal to adluminal compartment. As such, Sertoli-germ cell actin-based adherens junctions (AJ), such as ectoplasmic specializations (ES), must disassemble and reassemble to facilitate this event. Recent studies have shown that AJ dynamics are regulated by intricate interactions between AJ integral membrane proteins (e.g., cadherins, alpha6beta1 integrins and nectins), phosphatases, kinases, adaptors, and the underlying cytoskeleton network. For instance, the myotubularin (MTM) phosphoinositide (PI) phosphatases, such as MTM related protein 2 (MTMR2), can form a functional complex with c-Src (a non-receptor protein tyrosine kinase). In turn, this phosphatase/kinase complex associates with beta-catenin, a constituent of the N-cadherin/beta-catenin functional unit at the AJ site. This MTMR2-c-Src-beta-catenin complex apparently regulates the phosphorylation status of beta-catenin, which determines cell adhesive function conferred by the cadherin-catenin protein complex in the seminiferous epithelium. In this review, we discuss the current status of research on selected phosphatases and kinases, and how these proteins potentially interact with adaptors at AJ in the seminiferous epithelium to regulate cell adhesion in the testis. Specific research areas that are open for further investigation are also highlighted.     

Myotubularin-Related (MTMR) Phospholipid Phosphatase Proteins in the Peripheral Nervous System

Myotubularin-related proteins (MTMRs) constitute a broad family of ubiquitously expressed phosphatases with 14 members in humans, of which eight are catalytically active phosphatases, while six are catalytically inactive. Active MTMRs possess 3-phosphatase activity toward both PtdIns3P and PtdIns(3, 5)P ₂ poliphosphoinositides (PPIn), suggesting an involvement in intracellular trafficking and membrane homeostasis. Among MTMRs, catalytically active MTMR2 and inactive MTMR13 have a nonredundant function in nerve. Loss of either MTMR2 or MTMR13 causes Charcot–Marie–Tooth type 4B1 and B2 neuropathy, respectively, characterized by demyelination and redundant loops of myelin known as myelin outfoldings. In Mtmr2-null mouse nerves, these aberrant foldings occur at 3–4 weeks after birth, a time when myelination is established, and Schwann cells are still elongating to reach the final internodal length. Moreover, Mtmr2-specific ablation in Schwann cells is both sufficient and necessary to provoke CMT4B1 with myelin outfoldings. MTMR2 phospholipid phosphatase might regulate intracellular trafficking events and membrane homeostasis in Schwann cells during postnatal nerve development. In this review, we will discuss recent findings on the MTMR family with a major focus on MTMR2 and MTMR13 and their putative role in Schwann cell biology.     

Differential redox regulation within the PTP superfamily

The Protein Tyrosine Phosphatase (PTP) family comprises a large and diverse group of enzymes, regulating a range of biological processes through de-phosphorylation of many proteins and lipids. These enzymes share a catalytic mechanism that requires a reduced and reactive cysteine nucleophile, making them potentially sensitive to inactivation and regulation by oxidation. Analysis of ten PTPs identified substantial differences in the sensitivity of these enzymes to oxidation in vitro. More detailed experiments confirmed the following rank order of sensitivity: PTEN and Sac1>PTPL1/FAP-1>myotubularins. When the apparent sensitivity to oxidation of these PTPs in cells treated with hydrogen peroxide was analysed, this correlated well with the observed sensitivities to oxidation in vitro. These data suggested that different PTPs may fall into at least three different classes with respect to mechanisms of cellular redox regulation. 1. PTEN and Sac1 were readily and reversibly oxidised in vitro and in cells treated with hydrogen peroxide 2. PTPL1 appeared to be resistant to oxidation in cells, correlating with its sensitivity to reduction by glutathione in vitro 3. The myotubularin family of lipid phosphatases was almost completely resistant to oxidation in vitro and in cells. Our results show that sensitivity to reversible oxidation is not a necessary characteristic of the PTPs and imply that such sensitivity has evolved as a regulatory mechanism for some of this large family, but not others.     

Combinatorial control of the specificity of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs), the enzymes that dephosphorylate tyrosyl phosphoproteins, were initially believed to be few in number and serve a 'housekeeping' role in signal transduction. Recent work indicates that this is totally incorrect. Instead, PTPs comprise a large superfamily whose members play critical roles in a wide variety of cellular processes. Moreover, PTPs exhibit exquisite substrate specificity in vivo. Recent evidence has led us to propose that members of the PTP family achieve selectivity through different combinations of specific targeting strategies and intrinsic catalytic domain specificity.     

Bicyclic benzofuran and indole-based salicylic acids as protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) constitute a large and structurally diverse family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. Malfunction of PTP activity has significant implications in many human diseases, and the PTP protein family provides an exciting array of validated diabetes/obesity (PTP1B), oncology (SHP2), autoimmunity (Lyp), and infectious disease (mPTPB) targets. However, despite the fact that PTPs have been garnering attention as novel therapeutic targets, they remain largely an untapped resource. The main challenges facing drug developers by the PTPs are inhibitor specificity and bioavailability. Work over the last ten years has demonstrated that it is feasible to develop potent and selective inhibitors for individual members of the PTP family by tethering together small ligands that can simultaneously occupy both the active site and unique nearby peripheral binding sites. Recent results with the bicyclic salicylic acid pharmacophores indicate that the new chemistry platform may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Structural analysis of PTP-inhibitor complexes reveals molecular determinants important for the development of more potent and selective PTP inhibitors, thus offering hope in the medicinal chemistry of a largely unexploited protein class with a wealth of attractive drug targets.     

A novel phosphatase family, structurally related to dual-specificity phosphatases, that displays unique amino acid sequence and substrate specificity

Members of the superfamily of protein tyrosine phosphatases (PTPs) share the presence of an evolutionarily conserved PTP catalytic domain. Among them, the dual-specificity phosphatases (DSPs) constitute a diverse group of enzymes in terms of substrate specificity, including nonprotein substrates. In recent years, an increasing number of novel DSPs, whose functions and biological substrates are not well defined, have been discovered in a variety of organisms. In this study, we define the structural and functional properties of evolutionarily related atypical DSPs from different phyla. Sets of conserved motifs were defined that (i) uniquely segregated mammalian atypical DSPs from closely related enzymes and (ii) exclusively characterised a novel family of atypical DSPs present in plants, fungi, and kinetoplastids [plant and fungi atypical (PFA)-DSPs]; despite having different sequence “fingerprints,” the PTP tertiary structure of PFA-DSPs is conserved. Analysis of the catalytic properties of PFA-DSPs suggests the existence of a unique substrate specificity for these enzymes. Our findings predict characteristic functional motifs for the diverse members of the DSP families of PTPs and provide insights into the functional properties of DSPs of unknown function.