Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts

One type of membrane microdomain, enriched in glycosphingolipids and cholesterol and referred to as lipid rafts, has been implicated in the generation of activating signals triggered by a variety of stimuli. Several laboratories, including ours, have recently demonstrated that the B cell receptor (BCR) inducibly localizes to the rafts upon activation and that functional lipid rafts are important for BCR-mediated "positive" signaling. In the later phases of the immune response, coligation of the BCR and the inhibitory receptor Fc gamma RIIB1 leads to potent inhibition of BCR-induced positive signaling through the recruitment of the inositol phosphatase SHIP to Fc gamma RIIB1. One potential model is that the Fc gamma RIIB1 itself might be excluded from the rafts basally and that destabilization of raft-dependent BCR signaling might be part of the mechanism for the Fc gamma RIIB1-mediated negative regulation. We tested this hypothesis and observed that preventing BCR raft localization is not the mechanism for this inhibition. Surprisingly, a fraction of Fc gamma RIIB1 is constitutively localized in the rafts and increases further after BCR + FcR coligation. SHIP is actively recruited to lipid rafts under negative stimulation conditions, and the majority of Fc gamma RIIB1-SHIP complexes localize to lipid rafts compared with non-raft regions of the plasma membrane. This suggested that this negative feedback loop is also initiated in the lipid rafts. Despite its basal localization to the rafts, Fc gamma RIIB1 did not become phosphorylated after BCR alone cross-linking and did not colocalize with the BCR that moves to rafts upon BCR engagement alone (positive signaling conditions), perhaps suggesting the existence of different subsets of rafts. Taken together, these data suggest that lipid rafts play a role in both the positive signaling via the BCR as well as the inhibitory signaling through Fc gamma RIIB1/SHIP.     

Use of antisense oligonucleotides in functional genomics and target validation

With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.     

Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.     

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity

p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers.     

"Recruitment signals" from apoptotic cells: invitation to a quiet meal

An evolutionarily conserved machinery exists for engulfment of apoptotic cells from worm to mammals. New observations suggest that corpse clearance is tightly linked to apoptosis and that dying cells use both recruitment and eat-me signals for phagocyte attraction and recognition.     

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.     

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.