Biochemistry and Pharmacology - Research Publications
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ItemExtinction of a cocaine-taking context that protects against drug-primed reinstatement is dependent on the metabotropic glutamate 5 receptor(WILEY, 2015-05-01)We investigated the effects of extinguishing action-reward versus context-reward associations on drug-primed reinstatement, and the potential role of the metabotropic glutamate 5 receptor (mGlu5) in these different types of extinction in rats that self-administer cocaine. We observed that daily context extinction (non-reinforced exposures to the cocaine-taking context with retracted levers) was just as effective as daily lever extinction in reducing cocaine-primed reinstatement compared with passive abstinence. Additionally, systemic injections of the mGlu5 negative allosteric modulator MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine) following each extinction session significantly impaired the ability of context extinction to reduce cocaine-primed reinstatement, without affecting reinstatement after lever extinction or passive abstinence.
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ItemIn silico prediction of antimalarial drug target candidates(ELSEVIER SCI LTD, 2012-12-01)The need for new antimalarials is persistent due to the emergence of drug resistant parasites. Here we aim to identify new drug targets in Plasmodium falciparum by phylogenomics among the Plasmodium spp. and comparative genomics to Homo sapiens. The proposed target discovery pipeline is largely independent of experimental data and based on the assumption that P. falciparum proteins are likely to be essential if (i) there are no similar proteins in the same proteome and (ii) they are highly conserved across the malaria parasites of mammals. This hypothesis was tested using sequenced Saccharomycetaceae species as a touchstone. Consecutive filters narrowed down the potential target space of P. falciparum to proteins that are likely to be essential, matchless in the human proteome, expressed in the blood stages of the parasite, and amenable to small molecule inhibition. The final set of 40 candidate drug targets was significantly enriched in essential proteins and comprised proven targets (e.g. dihydropteroate synthetase or enzymes of the non-mevalonate pathway), targets currently under investigation (e.g. calcium-dependent protein kinases), and new candidates of potential interest such as phosphomannose isomerase, phosphoenolpyruvate carboxylase, signaling components, and transporters. The targets were prioritized based on druggability indices and on the availability of in vitro assays. Potential inhibitors were inferred from similarity to known targets of other disease systems. The identified candidates from P. falciparum provide insight into biochemical peculiarities and vulnerable points of the malaria parasite and might serve as starting points for rational drug discovery.
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ItemP2X7 Receptor-mediated Scavenger Activity of Mononuclear Phagocytes toward Non-opsonized Particles and Apoptotic Cells Is Inhibited by Serum Glycoproteins but Remains Active in Cerebrospinal Fluid(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-05-18)Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells.
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ItemThe Minimal Active Structure of Human Relaxin-2(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-10-28)H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.
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ItemOpposing Actions of Extracellular Signal-regulated Kinase (ERK) and Signal Transducer and Activator of Transcription 3 (STAT3) in Regulating Microtubule Stabilization during Cardiac Hypertrophy(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-01-14)Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.
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ItemBiosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-03-09)To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.
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ItemConservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-06-25)Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.
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Itemc-Jun N-terminal Kinase Phosphorylation of Stathmin Confers Protection against Cellular Stress(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-09-10)The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes.
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ItemAnionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-10-15)Although the N terminus of the prion protein (PrP(C)) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23-110) and N2 (23-89) fragments derived from constitutive processing of PrP(C) and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.
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ItemAromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology(ELSEVIER, 2009-07-01)Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.