Alumni Dissertations

 

Alumni Dissertations

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  • Exploration of Aporphines as MDMA and AChE Inhibitors

    Author:
    Stevan Pecic
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Wayne Harding
    Abstract:

    PART A MDMA (3,4-methylenedioxy methamphetamine) is a psychoactive drug which is thought to act via stimulation of secretion as well as inhibition of re-uptake of large amounts of serotonin, noradrenaline and dopamine in the brain. MDMA also acts directly on a number of receptors, including 5-HT2A receptors. There is considerable evidence that 5-HT2A antagonists can modulate behavioral and physiological effects of MDMA in animals. Nantenine an aporphine alkaloid ex Nandina domestica has been reported to block and reverse a range of behavioral and physiological effects of MDMA in mice. It is known that nantenine has moderate 5-HT2A antagonist activity. However, very little structure-activity relationship (SAR) studies have been performed on nantenine with regards to its activity at the 5-HT2A receptor. As part of our research focus to develop aporphine-based 5-HT2A antagonists as potential MDMA antagonists, we have prepared a library of novel analogs to investigate the structural requirements for nantenine's 5-HT2A activity. Our studies demonstrate that the N-methyl group, methylenedioxy ring and structural rigidity of the aporphine nucleus are important for activity, and that appropriate substitutions on the aromatic aporphine core can improve 5-HT2A antagonist activity. To elucidate possible binding modes of these compounds and to determine the correlation with our binding data, we built a 5-HT2A homology model based on a bovine rhodopsin template and then performed docking/scoring experiments. Our results suggest that members of the C1 series of nantenine analogs bind to the 5-HT2A receptor in the same orientation but differently than nantenine. In addition to known and important interaction between the protonated nitrogen of the ligands and Asp155 of the receptor, some of these analogs established a hydrogen bond with Ser242 as well as hydrophobic interactions with Phe234 and Gly238. These latter interactions may account for their enhanced activity as compared to nantenine. Our findings will be useful in the future design of high affinity 5-HT2A ligands based on the nantenine aporphine core structure. PART B For the second part of the project, nantenine as well as a number of flexible analogs were evaluated for acetylcholinesterase (AChE) inhibitory activity in a microplate spectrophotometric assays based on Ellman's method. It was found that the rigid aporphine core of nantenine and N-methyl substituent are important structural requirements for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors; inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aβ a major causative factor in the progression of Alzheimer's disease (AD).

  • ROLES OF IGH INTRONIC ENHANCER E&mu IN CLONAL SELECTION AT THE PRE-B TO IMMATURE B CELL TRANSITION AND IN THE ELIMINATION OF AUTOREACTIVE B CELLS

    Author:
    Cheng Peng
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Laurel Eckhardt
    Abstract:

    The immunoglobulin heavy chain locus (Igh) intronic enhancer, E&mu, enhances transcription of recombined Igh genes. We have previously shown that in mice with an E&mu-deficient Igh allele (VH&deltaa), Ig&mu is expressed at half of the wild-type levels in pre-B cells. We also described an E&mu-dependent "check-point", operating at the pre-B to immature B cell transition, for heavy chain allelic exclusion. We now show that deletion of E&mu results in a smaller immature B cell compartment, and the pre-BCR/BCR signaling is diminished in pre-B cells as a result of the reduced Ig&mu levels, making it difficult for emerging BCRs to reach the signaling threshold required for positive selection of pre-B cells to the immature B cell stage. Our hypothesis is that, to circumvent the problem of inadequate signaling, E&mu-deficient B cells either 1) expand the rare precursor B cells seemingly breaking the rules of allelic exclusion to express a second IgH allele as "double-producers", to achieve higher levels of Ig&mu-chain and hence higher pre-BCR and BCR levels, or 2) undergo heightened light-chain editing to create an IgH/IgL combination with superior signaling properties to make up for the lower Ig&mu-chain (lower BCR) levels and signaling. To test these hypotheses and to determine whether escape from the developmental defects in E&mu-deficient B cells is dependent upon light chain, we provided the E&mu-deficient mice with a pre-assembled VL gene (3-83V&kappa). This led to not only a larger immature B cell compartment, but also a decrease in "double-producers". We suggest that an IgH/IgL combination with superior signaling properties may compensate for the reduced BCR levels and eliminate the selective advantage of "double-producers". We also find that "double-producers" in E&mu-deficient heterozygous mice (VH&deltaa/WTb), include a subpopulation with autoreactive BCRs. We infer that the BCRs with IgH from the VH&deltaa allele are ignored during negative selection at the pre-B to immature B cell transition, due to their low density. Instead, the double-producers are both positively and negatively selected on the basis of BCRs with IgH from the alternate allele (with E&mu intact). Taken together, these results suggest that E&mu functions to ensure sufficient Ig&mu(IgH) levels at the pre-B to immature B transition, which has an important impact on the maintenance of heavy chain allelic exclusion, the breadth of the BCR repertoire, and the elimination of autoreactive B cells.

  • CHARACTERIZATION OF PAXILLIN, PHOSPHOLIPASE D AND THEIR FUNCTIONAL INTERACTION

    Author:
    Jelena Pribic
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Derrick Brazill
    Abstract:

    The actin cytoskeleton plays a fundamental role in various processes including differentiation, migration, endocytosis and exocytosis. An adapter protein, paxillin, as well as an enzyme, Phospholipase D (PLD), have been associated with processes based on actin cytoskeleton regulation. Such regulation is critical for the development of Dictyostelium discoideum. To gain better insight into the roles of paxillin and PLD and to investigate their potential interactions, we study the paxillin and PLD orthologs, PaxB and PldB, respectively. Previous work showed that in Dictyostelium discoideum, paxillin (PaxB) and Phospholipase D (PldB) colocalize and co-immunoprecipitate, suggesting that they physically interact. We found that the phenotypes observed during development, cell sorting and several actin-required processes including cAMP chemotaxis, cell-substrate adhesion, actin polymerization, phagocytosis, and exocytosis reveal a genetic interaction between paxB and pldB suggesting a functional interaction between gene products. Taken together, our data point to PldB being a required binding partner of PaxB during processes involving actin eorganization. Based on our study in the model organism Dictyostelium discoideum, we examined whether a similar relationship between paxillin and PLD exists in the highly aggressive human breast cancer cell line MDA-MB-231. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK complex. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, our results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, paxillin, FAK and Erk participate in the same signaling pathway in this breast cancer cell line. The proposed studies will allow further insight into the role of these proteins in cancer and better understanding of the clinical course of disease.

  • SEROTONIN 1A RECEPTOR MEDIATED NEUROGENESIS IN THE DEVELOPING HIPPOCAMPUS

    Author:
    Buddima Ranasinghe
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Probal Banerjee
    Abstract:

    The importance of the brain serotonin 1A receptor (5-HT1A-R) during postnatal brain development has been established, but the mechanism of its action in brain neurons remains unclear. It is currently known that the 5-HT1A-R plays a crucial role in the brain by regulating mood and behavior and 5-HT1A-R stimulation in adult mice has been suggested to induce neurogenesis in the adult neurogenic niches such as the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. In mice, absence of the 5-HT1A-R during development results in heightened anxiety and depressive behavior. However, the 5-HT1A-R mediated signaling activity that is responsible for its role during development is unknown. Among the hippocampal signaling pathways stimulated by the agonist-bound 5-HT1A-R, the mitogen activated protein kinase (MAPK) pathway is an important regulator of both division and survival of neuronal cells in the brain. Additionally, the Protein Kinase C (PKC) isozyme PKCɛ is an important signaling molecule that is highly expressed during early postnatal brain development particularly postnatal day 2-6 (P2-P6). Here we show that neurogenesis in the developing hippocampus at P6 in mice is dependent on both MAPK and PKCɛ. Our initial experiments use pharmacological inhibitors to confirm that PKCɛ mediates 5-HT1A-R-linked activation of MAPK. We then demonstrate that neurogenesis is increased upon stimulation of this 5-HT1A-R→PKCɛ→MAPK pathway both in a hippocampal-derived neuronal cell line stably expressing the 5-HT1A-R (HN2-5) and in the DG of P6 mice. Further, inhibition of either MAPK or PKCɛ considerably disrupts the burst in bromo-deoxy-uridine (BrdU) labeling and Ki-67 staining, showing neuroblast number, in the DG. As for a downstream signal that relays the proliferative signal from MAPK, we have identified the Retinoblastoma protein (Rb) as a potential target of MAPK, and shown that its phosphorylation dynamics may be tightly regulated in response to 5-HT1A-R stimulation. Therefore, our findings reveal a novel pathway involving PKCɛ, MAPK, and Rb through which the 5-HT1A-R potentially regulates neurogenesis during early postnatal hippocampal development.

  • DYNAMICS ON MULTIPLE TIMESCALES IN THE CYSTOVIRAL RNA-DIRECTED RNA POLYMERASE

    Author:
    Zhen Ren
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Ranajeet Ghose
    Abstract:

    The RNA-dependent RNA polymerase P2 from cystovirus ϕ6 directs the replication and transcription of the double-stranded RNA genomes. In spite of the availability of multiple crystal structures at various points along its catalytic pathway, the dynamics role involving in the catalytic cycle and fidelity control remain unclear. Isoleucine residues are distributed over the three-dimensional fold of P2. By using the δ1 positions of 25 Ile residues as probes, we measured the dynamic modes and their alterations along P2 catalytic cycle with CPMG-based multiple quantum relaxation dispersion experiments. The results indicate that P2 is dynamic on the fast (ps-ns) and slow (μs-ms) timescale. The characteristics of several motional modes are altered in the presence of substrate analogs and single-stranded RNA templates. The timescale of the lower frequency motional modes that involve several conserved functional motifs coincides with the catalytic timescale (1-2 ms), which was determined from kinetic analyses of representative RdRPs. We further investigated the influence of the extreme 3'-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on the slow motional modes using multiple-quantum relaxation dispersion. We found that P2, in the presence of templates bearing the proper genomic 3'-ends or the preferred initiation nucleotide (GTP), displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This further suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.

  • MOLECULAR INTERACTIONS OF ADIPOCYTE FATTY ACID-BINDING PROTEIN WITH ACTIVATING AND NON-ACTIVATING LIGANDS: PROTEIN OLIGOMERIZATION AND LIGAND BINDING SITES

    Author:
    Samar Rizk
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Ruth Stark
    Abstract:

    Intracellular lipid binding proteins involved in fatty acid transport and metabolism include adipocyte fatty acid-binding protein (AFABP), a 15 kDa polypeptide that plays a central role in the development of diabetes and atherosclerotic cardiovascular disease in experimental animals; the significant degree to which the protein is released into the bloodstream is thought to predict the development of Metabolic Syndrome. Upon binding of activating ligands such as linoleate and troglitazone (TDZ) or inactivating ligands such as oleate, AFABP has been proposed to adopt two alternative modes of self-association that activate or deactivate a nuclear localization signal. The goal of this study is to develop a molecular rationale for these contrasting ligand-associated signals. Both apo and liganded AFABP proteins were shown to maintain an overwhelmingly monomeric form in solution using size exclusion chromatography and static light scattering methods. Multidimensional solution-state nuclear magnetic resonance experiments were used to make sequential resonance assignments of the polypeptide backbone 1H and 15N nuclei. These assignments made possible ligand titration experiments that identified the key protein residues involved in the binding and the defined binding site. Comparative analysis of the binding sites in the three holo proteins demonstrated that oleate and linoleate bind similarly in a U-shaped configuration within the protein binding cavity despite their contrasting functional behavior, whereas the activating linoleate and TDZ ligands bind at dissimilar sites with the AFABP protein.

  • Metabolic Checkpoints in Cancer Cell Cycle

    Author:
    Mahesh Saqcena
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    David Foster
    Abstract:

    Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from GF signaling is one of the hallmarks in cancer; however, cancer cells also have metabolic rewiring enabling them to engage in anabolic biosynthetic pathways. In the absence of GF instructions and nutrients, cells commonly undergo apoptotic cell death. Thus, it is of importance to elucidate the differences between GF and nutrient deregulation in cancer to develop novel strategies in targeting tumor cell proliferation and survival. Here, we report that the GF-mediated mid-G1 restriction point (R) is distinct and distinguishable from a series of late-G1 metabolic checkpoints mediated by essential amino acids, conditionally essential amino acid - glutamine, and mTOR - the mammalian target of rapamycin. Our data indicate that the arrest sites mediated by various blocking conditions are in the order of GF -> EAA -> Q -> mTOR. We temporally mapped the EAA and glutamine checkpoints at 12 hr from G0 and mTOR mediated arrest occurring at 16 hr from G0. Distinct profiles for cell cycle regulator expression and phosphorylation was observed when released from restriction point relative to the metabolic checkpoints. These data are consistent with a mid-G1 R where cells decide whether they should divide, followed by late-G1 metabolic checkpoints where cells determine whether they have sufficient nutrients to divide. Since mTOR inhibition using rapamycin or Torin1 arrested the cells latest in G1, mTOR may serve as the final arbiter for nutrient sufficiency prior to replicating the genome. Significantly we also observed that in addition to GF autonomy, several cancer cells also have dysregulated nutritional sensing, and arrest in S- and G2/M phase upon essential amino acid and glutamine deprivation. We identified K-Ras mutation as the underlying genetic cause for this phenomenon. We found that treating cancer cells harboring K-Ras mutation with aminooxyacetate (AOA) - drug that interferes with glutamine utilization - causes them to arrest in S- and G2/M-phase, where synthetic lethality could be created to phase-specific cytotoxic drugs. Thus, besides addressing the long standing assumption of GF and nutrients regulating G1 cell cycle progression, our work provides rationale and proof of principle for targeting metabolic deregulations in cancer cells.

  • A TALE OF TWO NUTRITIONAL TARGETS: STUDIES OF MOLECULAR STRUCTURE AND PEDAGOGY

    Author:
    SAYANTANI SARKAR
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    RUTH STARK
    Abstract:

    Nutrition is one of the allied branches of biochemistry. Numerous nutritional targets in lipid metabolism and energy homeostasis are widely studied in biochemical research. For instance, liver fatty acid-binding protein is an important player in lipid metabolism. In this dissertation, two nutritional targets: physiological liver fatty acid-binding protein (LFABP) and natural tomato were studied from basic research and pedagogical perspectives. Three different projects were undertaken involving LFABP and tomato. (1) In the first project, solution state NMR was used to investigate ligand binding to LFABP. Oleate and linoleate exhibited moderately different binding locations in spite of apparently similar binding stoichiometries to LFABP. Additionally, oleate liganded holo LFABP showed chemical shift perturbations in presence of the anticoagulant drug warfarin, indicative of possible competition between oleate and warfarin as ligands of LFABP. However, reported claims of glucose and phytanic acid as ligands of LFABP could not be validated by solution state NMR. (2) In the second project, the basic research protocol concerning LFABP inspired a redesign of the City College undergraduate biochemistry laboratory course with an aim to incorporate a "research-inspired" module involving a new in-silico exercise. (3) In the third project, basic physical and spectroscopic research techniques used to study biopolymers in tomato were used to develop an adaptable suite of chemistry experiments for undergraduate and high school students. Both curricula were successfully tested on undergraduate and high school students in a cost effective fashion, demonstrating the feasibility of their implementation.

  • THE CATALYTIC MECHANISMS OF MYCOBACTERIUM TUBERCULOSIS CATALASE-PEROXIDASE (KATG) AND THE ORIGIN OF ANTIBIOTIC RESISTANCE IN THE KATG[S315G] MUTANT.

    Author:
    Javier Suarez
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Richard Magliozzo
    Abstract:

    Catalase-peroxidase (KatG) in M. tuberculosis is a bifunctional heme protein that exhibits both high catalase activity (2H2O2 → 2H2O + O2) and a broad-spectrum peroxidase activity (2AH + H2O2 → 2A* + 2H2O) and is responsible for activation of isoniazid (INH), a pro-drug used to treat TB infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. Nevertheless, there is a big gap in the M. tuberculosis literature with respect to the molecular origins of isoniazid resistance due to mutations in KatG. Here, we examined the origin of INH resistance caused by the KatG[S315G] mutant enzyme. Overexpressed KatG[S315G] was characterized by optical, EPR and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. INH resistance is suggested to arise from a redirection of catalytic heme intermediates into non-productive reactions that interfere with oxidation of INH. Previous studies have shown the formation of amino acid based radicals in KatG upon reaction with alkyl peroxide. However, the location and the possible function of these radicals are far from being resolved. In this study we tried to gain insights into the loci of radical formation through the analysis of cross-linking during turnover of KatG in the presence and absence of reducing substrate. SDS and Native-PAGE of KatG treated with peracetic acid or hydrogen peroxide under a variety of conditions demonstrate oligomers of molecular weight greater than that of the native dimer. The results are consistent with the hypothesis that cross-linking of KatG can occur in the absence of peroxidase substrates and that under physiological conditions, the activation of INH as well as the stability of KatG may be altered by this process. One of the most interesting structural features of Mtb KatG is the post-translational modification of residues Met 255, Tyr229 and Trp107, the side chains of which form an adduct on the distal side of the heme. Mutation of any of these three residues completely abolishes the catalase activity of KatG. A mechanism accounting for the robust catalase activity and the function of this adduct in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. Here, optical stopped-flow spectrophotometry, rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) spectroscopy both at X-band and at D-band, and mutagenesis were used to identify catalase reaction intermediates in Mtb KatG. Using rapid-freeze-quench EPR at X-band under catalase activity conditions (excess H2O2), a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical persists in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, abolishes this radical and the catalase activity. Therefore, a catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

  • Role of mammalian ubiquitin ligases UBR1 and UBR2 in cytosolic protein quality control

    Author:
    MST. RASHEDA SULTANA
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Avrom Caplan
    Abstract:

    UBR1 and UBR2 ubiquitin ligases function in the N-end rule degradation pathway in lower and higher eukaryotic cells. In yeast, the Ubr1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated via stress or with Hsp90 inhibitor GA. Based on these studies I examined the role of mammalian UBR1 in the degradation of protein kinase clients upon Hsp90 inhibition. I provide evidence that mammalian UBR1 promotes protein kinase quality control and sensitizes the cells to Hsp90 inhibition. The UBR1 deleted MEF cells showed reduced degradation of several protein kinases in the presence of GA. My findings also showed that Akt, p-Akt and Cdk4 the Hsp90 client protein kinases are still degraded in mouse UBR1 -/- cells treated with GA, but their levels recovered within 12-18 hours, in contrast to the wild type cells. The same findings were observed for human BT474 breast cancer cells with knocked down UBR1 by shRNA. These findings correlate with increased induction of Hsp90 expression in the Ubr1-/- cells compared with wild type cells. In addition, deletion of UBR1 and UBR2 showed resistance in terms of cell viability compared to wild type cells in the presence of GA and PU-H71. I also observed a reduction of UBR1 protein levels in GA-treated MEF and BT474 cells, suggesting that UBR1 is an Hsp90 client. I propose the existance of a novel feedback loop, where UBR1 negatively controls Hsp90 expression, while Hsp90 controls UBR1 stability. Further studies with CHIP reveal that CHIP and UBR1 have some functional overlap with respect to their E3 activities while UBR1 also affects the function of the Hsp90 chaperone machinery. My studies with other Hsp90 clients showed that UBR1 promotes degradation of steroid hormone receptors GR and AR but not the ER- £\. Co-expression of rUBR1 with hGR led to reduce the levels of hGR in the presence and absence of GA. There is a direct correlation between increasing UBR1 concentration and decreasing GR levels. Further studies addressed the specificity of that function with analysis of hAR and hER-fÑ. In this case, there was a significant reduction of the hAR levels when UBR1 was overexpressed, even in the absence of GA. By contrast, similar experiments with transfected hER-£\ suggest that UBR1 does not play a similar role in the degradation of this receptor. My combined findings suggest that UBR1 acts specifically in the clearance of GR and AR but not in ER-fÑ. All of these findings suggest that UBR1 is involved in the cytosolic protein quality control in mammalian system and it also plays a role in determining the sensitivity of the cells to the Hsp90 inhibitors.