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)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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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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ItemIn silico prediction of antimalarial drug target candidates(ELSEVIER SCI LTD, 2012-12)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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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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ItemStructure-function analyses of two plant meso-diaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-05-24)meso-Diaminopimelate decarboxylase catalyzes the decarboxylation of meso-diaminopimelate, the final reaction in the diaminopimelate l-lysine biosynthetic pathway. It is the only known pyridoxal-5-phosphate-dependent decarboxylase that catalyzes the removal of a carboxyl group from a d-stereocenter. Currently, only prokaryotic orthologs have been kinetically and structurally characterized. Here, using complementation and kinetic analyses of enzymes recombinantly expressed in Escherichia coli, we have functionally tested two putative eukaryotic meso-diaminopimelate decarboxylase isoforms from the plant species Arabidopsis thaliana We confirm they are both functional meso-diaminopimelate decarboxylases, although with lower activities than those previously reported for bacterial orthologs. We also report in-depth X-ray crystallographic structural analyses of each isoform at 1.9 and 2.4 Å resolution. We have captured the enzyme structure of one isoform in an asymmetric configuration, with one ligand-bound monomer and the other in an apo-form. Analytical ultracentrifugation and small-angle X-ray scattering solution studies reveal that A. thaliana meso-diaminopimelate decarboxylase adopts a homodimeric assembly. On the basis of our structural analyses, we suggest a mechanism whereby molecular interactions within the active site transduce conformational changes to the active-site loop. These conformational differences are likely to influence catalytic activity in a way that could allow for d-stereocenter selectivity of the substrate meso-diaminopimelate to facilitate the synthesis of l-lysine. In summary, the A. thaliana gene loci At3g14390 and At5g11880 encode functional. meso-diaminopimelate decarboxylase enzymes whose structures provide clues to the stereochemical control of the decarboxylation reaction catalyzed by these eukaryotic proteins.
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ItemCell-Autonomous Regulation of Astrocyte Activation by the Circadian Clock Protein BMAL1(CELL PRESS, 2018-10-02)Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults and plays a critical role in brain health and disease. We report that the core circadian clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism and a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression in vitro and in vivo, mediated in part by suppression of glutathione-S-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a mechanism by which the circadian clock could influence many aspects of brain function and neurological disease.
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ItemThe trans-Golgi network is a major site for α-secretase processing of amyloid precursor protein in primary neurons(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-02-01)Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase-mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor-like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.
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ItemStimulating the Release of Exosomes Increases the Intercellular Transfer of Prions(ELSEVIER, 2016-03-04)Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.