Physical Sciences and Mathematics Commons^™

Characterization Of The Dimerization Domains On The Mannose-6-Phosphate/Insulin-Like Growth Factor Ii Receptor, Tyler Degener

Theses/Capstones/Creative Projects

The mannose-6-phosphate/insulin-like growth factor II (M6P/IGF2) receptor is a transmembrane protein known to sequester growth factors from the extracellular matrix. This behavior suggests a mechanism of tumor suppression. Structurally, the receptor’s extracellular region is segmented into 15 homologous repeats, which are divided further into 5 triplet domains, labelled 1-3, 4-6, 7-9, 10-12, and 13-15. What is notable about the triplets is their propensity to form dimers with triplets on a second M6P/IGF2 receptor. In fact, previous studies indicate that this protein functions optimally when dimerized. Thus, the purpose of this experiment is to characterize these domain interactions. Using a urea …

Unfolded Protein Response Activation Reduces Secretion And Extracellular Aggregation Of Amyloidogenic Immunoglobulin Light Chain, Christina B. Cooley, L. M. Ryno, L. Plate, G. J. Morgan, J. D. Hulleman, J. W. Kelly, R. L. Wiseman

Christina B Cooley

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was …

Small Molecule Proteostasis Regulators That Reprogram The Er To Reduce Extracellular Protein Aggregation, L. Plate, Christina B. Cooley, J. J. Chen, R. J. Paxman, C. M. Gallagher, F. Madoux, J. C. Genereux, W. Dobbs, D. Garza, T. P. Spicer, L. Scampavia, S. J. Brown, H. Rosen, E. T. Powers, P. Hodder, R. L. Wiseman, J. W. Kelly

Christina B Cooley

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy …

Beyond Cell Penetrating Peptides: Designed Molecular Transporters, P. A. Wender, Christina B. Cooley, E. I. Geihe

Christina B Cooley

Inspired originally by peptides that traverse biological barriers, research on molecular transporters has since identified the key structural requirements that govern cellular entry, leading to new, significantly more effective and more readily available agents. These new drug delivery systems enable or enhance cellular and tissue uptake, can be targeted and provide numerous additional advantages of significance in imaging, diagnostics and therapy.

Broadly Applicable Methodology For The Rapid And Dosable Small Molecule-Mediated Regulation Of Transcription Factors In Human Cells, M. D. Shoulders, L. M. Ryno, Christina B. Cooley, J. W. Kelly, R. L. Wiseman

Christina B Cooley

Direct and selective small molecule control of transcription factor activity is an appealing avenue for elucidating the cell biology mediated by transcriptional programs. However, pharmacologic tools to modulate transcription factor activity are scarce because transcription factors are not readily amenable to small molecule-mediated regulation. Moreover, existing genetic approaches to regulate transcription factors often lead to high nonphysiologic levels of transcriptional activation that significantly impair our ability to understand the functional implications of transcription factor activity. Herein, we demonstrate that small molecule-mediated conformational control of protein degradation is a generally applicable, chemical biological methodology to obtain small molecule-regulated transcription factors that …

Oligocarbonate Molecular Transporters: Oligomerization-Based Syntheses And Cell-Penetrating Studies, Christina B. Cooley, B. M. Trantow, F. Nederberg, M. K. Kiesewetter, J. L. Hedrick, R. M. Waymouth, P. A. Wender

Christina B Cooley

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-side chain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one-step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.

Developmental Changes In Pedunculopontine Nucleus (Ppn) Neurons, T. Kobayashi, C. Good, J. Biedermann, Christina B. Cooley, R. D. Skinner, E. E. García-Rill

Christina B Cooley

The developmental decrease in rapid-eye-movement (REM) sleep in man occurs between birth and after puberty. We hypothesize that if this decrease in REM sleep does not occur, lifelong increases in REM sleep drive may ensue. Such disorders are characterized by hypervigilance and sensory-gating deficits, such as are present in postpubertal onset disorders like schizophrenia, panic attacks (a form of anxiety disorder), and depression. The decrease in REM sleep in the rat occurs between 10 and 30 days of age. We studied changes in size and physiological properties of pedunculopontine nucleus (PPN) cells involved in the control of arousal, i.e., waking …

Transition State Interactions In A Promiscuous Enzyme: Sulfate And Phosphate Monoester Hydrolysis By Pseudomonas Aeruginosa Arylsulfatase, Bert Van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian Hollfelder

Chemistry and Biochemistry Faculty Publications

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS^_WT has a much less negative Brønsted coefficient (ß_{leaving group} ^obs-Enz=-0.33) than the uncatalyzed reaction (ß_{leaving group} ^obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. …

Evaluating Methods Of Obtaining Male Pheromone From Hymenochirus Sp. Using Analytical Chemistry, Vincent Wing-Kun Leung

University of the Pacific Theses and Dissertations

Male Hymenochirus sp. frogs are known to release pheromone that attracts females of the same species. Four methods for collecting secretions containing pheromone in Hymenochirus sp. were tested: norepinephrine injection, gonadotropin-releasing hormone injection, homogenization of gland tissue, and electrostimulation of the skin over the breeding gland area. The samples collected were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry. The HPLC chromatograph for the male norepinephrine sample contained a peak at 6.4 min that was not in the female norepinephrine sample HPLC chromatograph. The male norepinephrine sample mass spectrum had a peak of m/z 292.0 not in the female …

Synthesis And Conformational Studies Of Various Amides, Marcos Beltran-Sanchez

University of the Pacific Theses and Dissertations

In the past, aminocyclohexanol rings have been successfully utilized as pH-triggered molecular switches in various trans-2-aminocyclohexanol derivatives. By changing the groups on the amine nitrogen, these models provided a wide pH range in which a switch can occur. The pH-induced switch of conformation was monitored by 1H-NMR spectroscopy. The models were also incorporated into the bilayer membrane of liposome structures and tested for their ability to disrupt their membrane upon their conformational flip induced by a decrease in pH.

In this work, the amide bond has been studied as a molecular switch and various amide derivatives have been tested for …