No description available.

The pathophysiology, etiology, and treatment of schizophrenia remain elusive, but research is closing the gap. Schizophrenia globally affects less than 1% of the population and presents with positive, negative, and cognitive symptoms. As treatment for schizophrenia is not completely and meaningfully effective at treating all of the symptoms, without eliciting side effects, the current thesis aimed to evaluate a new drug candidate. PAOPA is a novel allosteric modulator that increases dopamine binding to the dopamine D2 receptor. It has previously shown positive findings in preventing and reversing behaviours proposed to model phenotypes of schizophrenia. However, it has not yet been tested to improve cognitive deficits in animal models, nor has its effects on other animals models been investigated. Lastly, its mechanism of action has not yet been comprehensively answered. In three separate studies, PAOPA was tested on ameliorating attentional deficits using the 5-choice serial reaction time task in an amphetamine model, deficits in novel objection recognition memory, sensorimotor gating, social interaction, and locomotor activity using a PCP model, and its effects on proteins regulating G protein-coupled receptors (GRK2 and arrestin-3), downstream signalling (ERK1 and ERK2), and synaptic vesicular control (synapsin II) were investigated. Although the sample sizes were too small to draw valid interpretations, the results suggested that PAOPA partially attenuated deficits in attention, novel object recognition memory, social interaction, sensorimotor gating, but not locomotor. Furthermore PAOPA increased the protein expression of GRK2, arrestin-3, ERK1 and 2, and synapsin IIa in the medial prefrontal cortex, striatum, and the nucleus accumbens. The results suggest that PAOPA influences the dopaminergic system in the striatum to change behaviour via receptor internalization and possibly downstream signalling. The present studies illuminate new insights, and point to future explorations for the potential development of PAOPA as a therapeutic for schizophrenia. / Thesis / Master of Science (MSc)

NMDA glutamate receptor is a ligand-gated ion channel that mediates a major component of excitatory neurotransmission in the central nervous system (CNS). NMDA receptors are activated by simultaneous binding of two different agonists, glutamate and glycine/ D-serine1. With aging, glutamate concentration gets altered, giving rise to glutamate toxicity that contributes to age-related pathologies like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and dementia88,95. Some treatments for these conditions include NMDA receptor blockers like memantine130. However, when completely blocking the receptors, there is a restriction of the receptor's normal physiological function59. A different approach to regulate NMDAR receptors is thorough allosteric modulators that could allow cell type or circuit-specific modulation, due to widely distributed GluN2 expression, without global NMDAR overactivation59,65,122. In one study, we hypothesized that the compound CNS4 selectively modulates NMDA diheteromeric receptors (GluN2A, GluN2B, GuN2C, and GluN2C) based on (three) different glutamate concentrations. Electrophysiological recordings carried out on recombinant NMDA receptors expressed in xenopus oocytes revealed that 30μM and 100μM of CNS4 potentiated ionic currents for the GluN2C and GluN2D subunits with 0.3μM Glu/100μM Gly. However, when using 300μM Glu/100μM Gly, CNS4 inhibited the relative response in the GluN2D subunit and had no effect on the remaining subunits. CNS4 reduced the response to glutamate alone for GluN2A but increased it for GluN2B and did not appear to replace glutamate. Another set of electrophysiological recordings measuring current-voltage relationship was made in order to understand ion flow across the channel in the presence of CNS4. 100μM CNS4 numerically increased the ionic inward current through the channel pore with more positive membrane potential, reflected by a significant difference in reversal potential values, in the GluN2C and GluN2D subunits. CNS4 also exhibited a non-voltage dependent activity and it did not appear to compete with magnesium which naturally blocks the receptor. Finally, the effect of CNS4 on calcium uptake and cellular viability was study in neurons from primary rat brain culture. Cortial and striatal neurons were given excessive doses of synthetic agonist NMDA in order to hyperactivate native NMDAR. In the calcium assay, 100µM of CNS4 significantly increased calcium upatake when given with 300µM NMDA compared with NMDA alone in cortex and when given with 100µM and 300µM NMDA in striatum. In the MTS assay, CNS4 did not alter neuronal viability in either cortical or striatal neurons compared with NMDA alone. Also, when CNS4 was used in non treated neurons it did not alter neuronal viability. Findings from the primary brain culture let us conclude that CNS4 could facilitate calcium influx and possibly be non toxic for neurons. / Master of Science / NMDA ionotropic glutamate receptors are predominately expressed in the central nervous system (CNS). These receptors are activated by glutamate and glycine/ D-serine1. With aging, glutamate concentration in the synapse gets altered giving rise to toxic environments for neurons that can contribute to age-related pathologies like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and dementia88,95. Some treatments of these conditions include the receptor blockers like memantine130. However, when completely blocking the receptors, there is a restriction of the receptor's normal physiological function59. A different approach to regulate NMDAR is through allosteric modulators that are compounds that modulate the receptor function without competing with endogenous agonists59,65,122. In this study, we hypothesized that the compound CNS4 selectively modulates NMDAR based on glutamate concentration. Electrophysiological recordings on stage four xenopus oocytes helped us to identify the dose-dependent activity of CNS4 and we found that 30 and 100μM of CNS4 selectively potentiates ionic currents for GluN2C and GluN2D subunits with 0.3μM Glu/100μM Gly but inhibited currents for only GluN2D with 300μM Glu/100μM Gly. Following this, a current-voltage plot was made to examine the channel activity of CNS4. We found a numerical increase of ionic inward current through the channel pore with more positive membrane potential values in the GluN2C and GluN2D subunits. Also, the effect of CNS4 on the ion current activity changed based on glutamate concentration, and CNS4 did not exhibit a voltage-dependent activity, which is a positive feature for compounds that target the receptor133. Finally, to better understand the effect of the compound CNS4 in primary neurons in a toxic environment, a rat brain neuronal culture was made. Increasing doses of NMDA with constant 100µM CNS4 increased cellular Ca2+ influx in a dose-dependent manner. Particularly, 100µM CNS4 with 300µM NMDA exhibited a significant increase in Ca2+ influx in both cortical and striatal neurons compared with 300µM NMDA alone. However, when used alone, 100µM CNS4 did not have an effect on the amount of Ca2+ influx. In addition, CNS4 plus NMDA did not increase viability compared to NMDA alone, and CNS4 alone did not proportionally reduce neuronal viability.

Alpha7 Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in essential physiological functions and play a role in disorders such as Alzheimer’s disease. MD-354 (3-chlorophenylguanidine; 21), the first small–molecule negative allosteric modulator (NAM) at alpha7 nAChRs, served as a lead in developing structure–activity relationships for NAMs at a7 nAChRs. MD-354 (21) also binds at 5-HT3 receptors. Analogs of MD-354 with structural features detrimental to 5-HT3 receptor affinity were evaluated in patch-clamp recordings and an aniline N-methyl analog resulted in a more potent and selective NAM than MD-354. A new N-methyl series of compounds was synthesized in which the 3-position was replaced with different substituents considering their electronic, lipophilic, and steric nature. Comparative studies were initiated to investigate whether or not the MD-354 series and the N-methyl series bind in the same manner; 3D models of the extracellular domain of human alpha7 nAChRs were developed, allosteric sites identified, and docking studies conducted.

α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in bacteria, including Neisseria meningitidis and Mycobacterium tuberculosis. It catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Like many key enzymes in biosynthesis, α-IPMS is inhibited by the end-product of the biosynthetic pathway, in this case leucine. α-IPMS is homodimeric, with monomers consisting of a (β/α)8-barrel catalytic domain, two subdomains and a C-terminal regulatory domain, responsible for binding leucine and providing feedback inhibition for leucine biosynthesis. The exact mechanism of feedback inhibition in this enzyme is unknown, despite the elucidation of crystal structures with and without leucine bound. This thesis explores the nature of allosteric regulation in α-IPMS, including the effects of the regulatory domain and the importance of structural asymmetry on catalytic activity. Chapter 2 details the characterisation of wild-type α-IPMS from N. meningitidis (NmeIPMS). This protein was successfully cloned, expressed and purified by metal-affinity and size-exclusion chromatography. NmeIPMS has similar characteristics to previously characterised α-IPMSs, being a dimer and demonstrating substrate binding affinities in the micromolar range. This enzyme has a turnover number of 13s⁻¹ and is sensitive to mixed, non-competitive inhibition by the amino acid leucine. Small angle X-ray scattering experiments reveal that the solution-phase structure of this protein is likely similar to existing crystal structures of other α-IPMSs. In Chapter 3, substitutions of residues potentially involved in the binding and transmission of the leucine regulatory mechanism are described. Most of these amino acid substituted variants reduce enzyme sensitivity to leucine, and one variant is almost entirely insensitive to this inhibitor. Another of these variants demonstrates an unexpected decrease in substrate affinity, despite the substituted residue being located far from the active site. The independence of α-IPMS domains is investigated in Chapter 4. The catalytic domains were isolated from NmeIPMS and the α-IPMS from M. tuberculosis (MtuIPMS), and found to be unable to catalyse the condensation of substrates, despite maintaining the wild-type structural fold. Complementation studies with Escherichia coli cells lacking the gene for α-IPMS show that the truncated variants are unable to rescue growth in these cells. Binding of α-KIV in the truncated NmeIPMS variant is much stronger than in the wild-type, and this may be the reason for lack of competent catalysis. A crystal structure was solved for the truncated variant of NmeIPMS and indicates that the regulatory domain is required for proper positioning of large regions of the protein. Two isolated regulatory domains from NmeIPMS were cloned, but with limited success in characterisation. Finally, Chapter 5 describes substitutions made in MtuIPMS to affect relative domain orientations within the protein. Dimer asymmetry is investigated by substituting residues at the domain interfaces. These substitutions did have some effect on catalysis and inhibition, but did not show any change in average solution-phase structure. These results are drawn together in the greater context of allostery in general in Chapter 6, along with ideas for future research in this field. This chapter reviews the insights gained into protein structure from this thesis, particularly the importance of residues at protein domain interfaces. The asymmetry in the α-IPMS structure is discussed, along with small-molecule binding regulatory domains.

No description available.

Call number: LD2668 .T4 1984 S87 / Master of Science

Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity.  The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR. Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively.  The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group. We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.

Thesis advisor: Evan R. Kantrowitz / E.coli Aspartate transcarbamoylase (ATCase) is the allosteric enzyme that catalyzes the committed step of the de novo pyrimidine biosynthesis pathway. ATCase facilitates the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The holoenzyme is a dodecamer, consisting of two trimers of catalytic chains, and three dimers of regulatory chains. ATCase is regulated homotropically by its substrates, and heterotropically by the nucleotides ATP, CTP, and UTP. These nucleotides bind to the regulatory chains, and alter the activity of the enzyme at the catalytic site. ATP activates the rate of ATCase's reaction, while CTP inhibits it. Additionally, UTP and CTP act together to inhibit the enzyme synergistically, each nucleotide enhancing the inhibitory effects of the other. Two classes of CTP binding sites have been observed, one class with a high affinity for CTP, and one with a low affinity. It has been theorized that the asymmetry of the binding sites is intrinsic to each of the three regulatory dimers. It has been hypothesized that the second observed class of CTP binding sites, are actually sites intended for UTP. To test this hypothesis, and to gain more information about heterotropic regulation of ATCase and signal transmission in allosteric enzymes, the construction of a hybrid regulatory dimer was proposed. In the successfully constructed hybrid, each of the three regulatory dimers in ATCase would contain one regulatory chain with compromised nucleotide binding. This project reports several attempts at constructing the proposed hybrid, but ultimately the hybrid enzyme was not attained. This project also reports preliminary work on the characterization of the catalytic chain mutant D141A. This residue is conserved in ATCase over a wide array of species, and thus was mutated in order to ascertain its significance. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Healthy cells continuously produce proteins to accomplish various functions, including immune responses, reaction catalyses, transmitting signals, structural supports and molecular transport. Protein needs to fold correctly into three-dimensional shape in order to function well, using the information stored in the amino acid sequence. Proteins may fold spontaneously in solution, but the situation in living cells can be complicated. Cells are filled with nucleic acids and proteins thus they are usually in a stressful environment. Under such circumstances, proteins can be unfolded or misfolded, leading to non-function or even toxicity. Cells employ molecular chaperones to solve protein folding problems. Among the many types of chaperones, heat shock proteins of approximately 70KDa (Hsp70s) act as a hub, because its functions feed into other members of the chaperone network. Hsp70s help to stabilize nascent polypeptides, facilitate cross-membrane translocation, refold the misfolded proteins, and guide non-recoverable denatured proteins to degradation. Hsp70s have explicit role in cancer cells, because elevated metabolism requires increased Hsp70s’ activity to avoid apoptosis and ensure survival. Hsp70s also help to prevent neurodegenerative diseases, and decreased level of Hsp70s is found in age-related symptoms and diseases. In general, it is well understood what Hsp70s can do, but little is known how Hsp70s do the job. Hsp70s are present and highly conserved in all living species, comprised of two structural domains. The nucleotide binding domain (NBD) binds and hydrolyzes ATP, while the substrate binding domain (SBD) binds and releases hydrophobic peptides. Although Hsp70s are known to act as an allosteric molecular machine, the details are elusive about how the domains are regulated. Besides, how nucleotide binding affects the Hsp70s’ function, and how ATP hydrolysis is performed are also unknown. In this thesis, I first introduce salient background on the Hsp70 subject, then explore previously unclear aspects of Hsp70 allosteric regulation and catalytic activity in two chapters describing my dissertation research, and finally conclude with my perspectives on future directions.