Molecular Biology and Biochemistry Electronic Theses and Dissertations (UMKC)
https://hdl.handle.net/10355/9982
The items in this collection are the scholarly output of UMKC graduate students.2024-03-28T21:38:40Z3-Hydroxy-3-methylglutaryl-coenzyme A lyase: investigation of cysteines mediating intersubunit disulfide formation and regulation by thiol/disulfide exchange and discovery of an extramitochondrial homolog
https://hdl.handle.net/10355/12492
3-Hydroxy-3-methylglutaryl-coenzyme A lyase: investigation of cysteines mediating intersubunit disulfide formation and regulation by thiol/disulfide exchange and discovery of an extramitochondrial homolog
Montgomery, Christa L. Cochran
3-Hydroxy-3-methylglutaryl-Coenzyme A lyase catalyzes the cleavage of 3-hydroxy-
3-methylglutaryl-Coenzyme A into acetoacetate and acetyl-Coenzyme A, a key reaction
in ketogenesis and leucine catabolism. Previous work prompted the hypothesis that in the
absence of reductant, cysteine 323 forms a disulfide bond with cysteine 323 on the
adjacent monomer, blocking the substrate's access to the active site which results in
diminished enzyme activity. The recently published crystal structure of the human
enzyme indicates that cysteines 323 on each monomer are too far apart to form a
disulfide. Each of the eight cysteines has been individually mutated to serine and the
mutants analyzed for activity and dimer formation in the presence and absence of
reductant. C170S, C266S, and C323S do not form dimers in the absence of reductant.
C170S, C197S, and C234S have an inflated dependence on thiol, C174S, C307S, and Therefore intersubunit disulfide bond formation does not directly correlate with
diminution of activity in the absence of thiol as previously proposed. Analysis of
C170S/C174S, C170S/C266S and C170S/C323S double mutants demonstrate that C170S
is not directly involved in the intersubunit disulfide bond and suggest that C170 may be
involved in regulation of activity by thiol/disulfide exchange. A C266S/C323S mutant
heterodimer restores covalent dimer formation confirming that these residues are
involved in intersubunit disulfide bond formation. Based on sequence homology, the
Mammalian Gene Collection Program identified the protein encoded by the gene 3-
hydroxy-3-methylglutaryl-Coenzyme A lyase-like 1 as being a potential 3-hydroxy-3-
methylglutaryl-Coenzyme A lyase. Characterization of the purified recombinant human
protein confirms that it is an authentic 3-hydroxy-3-methylglutaryl-Coenzyme A lyase.
In-vitro myristoylation experiments confirm that the protein is modified by Nmyristoyltransferase
and immunofluorescence microscopy in COSI cells demonstrate that
this modification effects the subcellular localization of the protein. Blots of rodent organ
lysates reveal expression primarily in the small intestine and that the protein is
overexpressed in human neuroblastoma and glioma cell lines compared to mouse brain. Co-localization experiments failed to determine to which compartment the lyase-like
protein is localized but confirm it is not mitochondrial or peroxisomal as is the traditional
lyase.
Title from PDF of title page, viewed on January 18, 2012; Dissertation advisor: Henry M. Miziorko; Vita; Includes bibliographic references (p. 81-89); Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2011
2012-01-18T00:00:00ZA single molecule study on the structural basis of ion selective permeation in voltage-gated sodium channels
https://hdl.handle.net/10355/86702
A single molecule study on the structural basis of ion selective permeation in voltage-gated sodium channels
Vance, Joshua
High resolution studies of voltage-gated sodium channels (VGSCs) can not reveal the dynamic nature of the channel structures, especially in lipid environments, which are vital for rational drug design. The bacterial NavAb channel is highly like its eukaryotic orthologs in structure, function, and pharmacological profiles, serving as an ideal model for biochemical characterizations. I purified the NavAb channel proteins and reconstituted them into liposomes. With liposome flux assays, I showed that the purified NavAb channels are highly conductive for Na+ and H+, but less permeable to K+, Cs+, and the bulky cation N-methyl-D-Glucamine. The NavAb channel is cysteine free, which allowed for introduction of cysteine mutations at different sites to be labeled with fluorophores. Thus, the structural dynamics of the NavAb channel can be examined utilizing single-molecule FRET. It is proposed that the ion selectivity of VGSCs is based on the size and charge, implying very minimized flexibilities in the selectivity filter. However, it was found that selectivity collapse underlies the slow inactivation in VGSC, including NavAb. To address the discrepancies, I examined the structural dynamics of the NavAb selectivity filter under different ionic conditions and membrane voltages. Surprisingly, my smFRET data showed large spontaneous FRET transitions which suggest significant structural dynamics at the NavAb selectivity filter. Moreover, I also detected significant FRET changes at the NavAb selectivity filter induced by activating voltage. My results indicate that ion selectivity in VGSC is not completely based on size and charge and needs to be investigated further.
Title from PDF of title page viewed September 9, 2021; Thesis advisor: Shizhen “Jeff” Wang; Vita; Includes bibliographical references (pages 37-40); Thesis (M.S.)--School of Biological and Chemical Sciences. University of Missouri--Kansas City, 2021
2021-01-01T00:00:00ZAlterations in cardiac contractility and heart rate mediated by bone loading
https://hdl.handle.net/10355/90323
Alterations in cardiac contractility and heart rate mediated by bone loading
Gray, Mark A
The skeleton is a dynamic organ in complex interplay with the other systems of the body and is highly responsive to input from the external environment. Increasingly, evidence is mounting that the skeleton is the source of endocrine factors which affect both long term cardiac outcomes and acute cardiac performance.
Some factors, like FGF23, have been shown to augment calcium handling in cardiomyocytes while at the same time being used clinically as a predictor of cardiomorbidity in, for example, chronic kidney disease patients. Other factors, such as osteocalcin, have been shown to be sufficient to elicit a type of cardiac stress response, even in adrenalectomized mice. The circulating levels of these and other bone-derived factors are known to respond to activity level and exercise. A major environmental input for the skeleton, especially during exercise, is movement-induced strain.
Media conditioned by MLO-Y4 osteocyte cell line culture under fluid flow sheer stress was used to model the acute effects of bone strain on the contraction magnitude and contraction rate of ex vivo Langendorf-perfused hearts. EKG was then used to measure cardiac parameters during tibial straining of anesthetized mice. A serum sample was collected after tibia strain and analyzed by LC/QToF MS. We found that MLO-Y4 conditioned media increased both the total force and the peak force of Langendorf-perfused hearts by approximately 25%. Somewhat paradoxically, in anesthetized mice we found that a 2-minute tibia strain induced a decrease in heart rate and an increase in heart rate variability which began within seconds, peaked after approximately one minute, then returned to baseline by the time tibia loading ended. LC/QToF was able to identify a variety of serum factors in the strained mice, which produced clusters when principle component analysis was used. One factor, which was the only factor statistically elevated in all groups, was increased in all individuals and had a molecular weight corresponding to acetate.
The results obtained in this study strongly suggests that the skeleton, responding to exercise-like mechanical strain, has the potential to rapidly augment cardiac performance. This may have implications in exercise training as well as reduced-function settings, such as bedrest, and may shed additional light on the interplay between bone and heart health.
Title from PDF of title page viewed June 3, 2022; Thesis advisor: Michael Wacker; Vita; Includes bibliographical references (pages 46-53); Thesis (M.S.)--School of Biological and Chemical Sciences. University of Missouri--Kansas City, 2022
2022-01-01T00:00:00ZAnalyzing the effects of Cis-elements and trans-factors on the stability of the Gal1 mRNP
https://hdl.handle.net/10355/45612
Analyzing the effects of Cis-elements and trans-factors on the stability of the Gal1 mRNP
Pirani, Karim
The highly dynamic and nonrandom spatial organization of the eukaryotic nucleus plays an important role in the regulation of gene expression. For example, in S. cerevisiae, several conditionally expressed genes relocate to the nuclear periphery upon activation. Moreover, these genes can be retained at the nuclear periphery for a considerable time after transcriptional shutoff. Sequence specific DNA binding proteins, transcription, chromatin remodeling, and mRNP quality control factors have all been implicated in perinuclear gene repositioning, but their relative contributions to the events of gene recruitment, capture and retention at the periphery remain unresolved. Sus1 is a conserved eukaryotic protein involved in transcription, mRNA export and perinuclear gene repositioning. Here, we show that the functions of Sus1p in perinuclear repositioning of GAL genes and its chromatin-linked functions can be genetically uncoupled, and that the role of Sus1p in the retention of mRNA in gene-proximal foci is chromatin-independent. Histone variant H2A.Z, likewise has been suggested to play a role in the post-transcriptional association of the yeast genes with the nuclear periphery. Our findings indicate that the loss of H2A.Z doesn’t alter gene-proximal mRNA retention, but has a differential effect on perinuclear repositioning of GAL genes.
H2A.Z affects recruitment of GAL1 promoter-driven genes to the nuclear periphery in a 3’UTR-dependent manner, but its effect on posttranscriptional retention of GAL1 genes at the nuclear periphery is locus specific. Our previous analysis has also shown that the retention of RNA at the site of transcription is dependent on 3’end of the Gal1. Our results indicate that the absence of AU-rich element (ARE) sequence in Gal1 3’UTR may be one of the factor leading to Gal1 mRNP formation at the transcription site. Moreover, our data shows that by introducing the ARE sequence (TATTTAT), between the two cleavage sites of Gal1 3’UTR, it not only reduces the number of cells that for Gal1 mRNP but also abolishes the synthesis of extended transcript at Gal1 locus.
Title from PDF of title page, viewed on June 8, 2015; Dissertation advisor: Michael O’Connor; Vita; Includes bibliographic references (pages 90-102); Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2014
2014-01-01T00:00:00Z