Thesis (Ph. D.)--University of Michigan.

Modern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and unnatural compounds that are structurally distinct from their target substrate. The widespread existence of such promiscuity could prove generally useful both in the natural and directed evolution of new proteins. To probe the persistence of enzyme promiscuity in modern proteomes we studied the model organism Escherichia coli due to its rapid growth, ease of genetic manipulation and many years of prior research on this organism which have generated abundant knowledge on its metabolism. The first exploration into uncovering enzyme promiscuity, described in chapter two, examines the proton transfer reaction catalyzed by triosephosphate isomerase (TIM). Triosephosphate isomerase catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the E. coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding for a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of L-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate. In chapter three we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a Kd value of 5.8 ± 0.2 µM in the absence of NADPH and a Kd value of 11 ± 1.3 µM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process. In chapter four we probe the network of genes within E. coli that can provide resistance to the nonnatural toxin bromoacetate. Microbial niches contain toxic chemicals that are capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival following exposure to a lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. Genetic experiments also identify 63 single-gene mutants of E. coli that display increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene that encodes a glutathione transferase capable of catalyzing the detoxification of bromoacetate with a kcat/Km value of 5.4 × 103 M-1 s-1. The catalytic proficiency of YliJ, which exceeds 5 orders of magnitude, is particularly noteworthy considering the enzyme is unlikely to have previously encountered bromoacetate. In total, our results indicate that nearly 2% of the E. coli proteome contributes to, or can be recruited to provide, bromoacetate resistance. This illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins. The work described here illuminates the vast metabolic and functional plasticity of protein function harbored within bacteria. Their ability to recruit latent and weakly active proteins for novel functions enables survival under diverse nutritional and environmental challenges. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / June 14, 2010. / Enzyme Function, Antibiotic Resistance, Glutathione Transferase / Includes bibliographical references. / Brian Miller, Professor Directing Dissertation; Hank Bass, University Representative; Hong Li, Committee Member; Lei Zhu, Committee Member; M. Elizabeth Stroupe, Committee Member.

This dissertation presents the concept, development, and characterization of a new methodology for both qualitative and quantitative analysis of protein digests in solution. Two beam fluorescence cross correlation spectroscopy is used to characterize the migration rates of fluorescently labeled peptides present in a poly (methyl methacrylate) (PMMA) microfluidic system. To achieve ultimate sensitivity, a two beam confocal microscope is employed to allow low background, single molecule detection. Two spatially separated laser beams are focused to near-diffraction limited spots and then positioned a few microns apart within a narrow region of a PMMA microdevice. Mobility measurements of the protein fragments are determined by the transit time for a single peptide to traverse through both detection volumes. Cross correlation of the fluorescence intensity signals from each confocal volume is used characterize the distribution of transit times. Electrophoresis conditions are employed and each peptide in a mixture will migrate at a characteristic velocity that depends on its size and charge. The cross correlation analysis yields a distribution of velocities reminiscent of an electropherogram in that each peak is evidence of an individual peptide. For a specific peptide digest, one can generate a fingerprint spectrum from the cross correlation data. The fingerprint could then be matched to a library of individual protein spectra allowing the rapid identification of the protein from whence the peptide mixture was derived. Our proposed method eliminates some of the shortcomings associated with current microfluidic technology. For example, analytes are monitored in free solution without actually separating the mixture; this eliminates the need for generating an analyte plug or migration over long distances. Also, since single molecule fluorescence is utilized it is possible to analyze multiple complex species at sub-nanomolar concentrations, in turn minimizing sample consumption. The two-beam fluorescence cross correlation method has the potential to be a high speed, highly sensitive alternative approach for protein and peptide analysis. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2006. / May 3, 2006. / Electrophoresis, Proteomics, Microfluidics, Single Molecule, Fluorescence / Includes bibliographical references. / Kenneth D. Weston, Professor Directing Dissertation; Peter G. Fajer, Outside Committee Member; Joseph B. Schlenoff, Committee Member; Oliver Steinbock, Committee Member.

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<p>The incidence and prevalence of people suffering from end stage renal disease is increasing. Proteinuria, particularly the presence of albumin in urine is concerning because proteinuria is associated with the progression to end stage renal disease (ESRD). Understanding the mechanisms involved in damaging the glomerular filtration barrier is essential. Transforming growth factor beta (TGFB) is a key cytokine in mediating glomerulosclerosis and proteinuria. Not much is known about the downstream pathways that mediates the renal damage and proteinuria.</p> <p>I hypothesize that TGFB induces proteinuria through podocyte de-differentiation and this occurs through SMAD dependent and independent pathways.</p> <p>Methods: I used adenovirus mediated gene transfer of TGFB1 to rat renal artery to study the effects of TGFB1 on renal structure and functions. To study the importance of SMAD3 in mediating downstream effects of TGFB1 in proteinuria and podocyte effacement, I used an anti-glomerular basement membrane model in SMAD3+/+ and SMAD3-/- mice to induce glomerulonephritis and proteinuria.</p> <p>Results: Transient TGFB1 overexpression via AdTGFB1 induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGFB1. TGFB1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. In cultured podocytes, TGFB1 downregulated the gene and protein expression of both nephrin and synaptopodin. These findings suggest that locally produced TGFB1 can cause podocyte de-differentiation marked by a loss of synaptopodin, nephrin, and foot process effacement; this process is partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.</p> <p>Both SMAD3+/+ and SMAD3-/- mice had proteinuria after induction of anti-GBM glomerulonephritis, though to a lesser extent in SMAD3-/- mice. SMAD3-/- and SMAD3+/+ mice developed significant glomerulonephritis with progressive interstitial fibrosis and chronic renal impairment. The SMAD3+/+ mice were found to be more prone to fibrotic changes, interstitial damage and tubular and glomerulosclerosis than the SMAD3-/- mice. This suggests that TGFB1 signals through pathways other than SMAD3 such as those triggered by hypoxia.</p> <p>Conclusion: I have shown that TGFB1 upregulation via AdTGFB1 induces proteinuria through podocyte dedifferentiation and FP effacement. Angiopoietins are essential for TGFB1 mediated podocyte injury. The effects of TGFB are partially mediated through SMAD3 as there is residual podocyte effacement and proteinuria in the SMAD3-/- mice. Hence there are SMAD3 dependent and independent pathways involved in proteinuria.</p> / Doctor of Science (PhD)

Axon degeneration can result from primary damage due to a variety of causes, and in some instances, its effects can further propagate damage to vicinal neurons. When an axon has been damaged or transected, Wallerian degeneration is the apoptotic-like mechanism that is initiated, ultimately leading to the death of the neuron. The post-injury cellular inflammatory response is recruited to clear the degraded debris and is also responsible for activating the cascade events leading to additional cell death in surrounding neurons. A unique strain of mutant mice were discovered to express what is called the Wallerian Degeneration Slow (Wld') gene, which produces a chimeric nuclear protein that has been observed to dramatically delay both the onset of axon degeneration and the initiation of the post-injury cellular inflammatory response . Recent studies seem _to indicate the neuroprotective phenotype induced by the Wld' protein is the result of it modulating levels of genetic and protein expression in the damaged neuron. This thesis will review what is known about the Wld' protein and discuss how it offers an in-depth look at the molecular mechanisms behind neurodegenerative injury and disease states. In addition, we will discuss our efforts of isolating and purifying plasmids with the Wld' gene that have been cloned in order to create a stable cell line to aid in the future study for the characterization of neuroprotective mechanisms and the molecular pathways of neurodegeneration .

<p> Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time.</p><p> Recent mechanistic studies have shown that PXR is modulated by multiple PTMs. In this thesis work, we conducted the first detailed examination of PXR regulation by acetylation. We found that PXR is efficiently acetylated <i> in vitro</i> and <i>in vivo</i> in multiple cell lines (293T, HepG2, LS174T). Acetylation and deacetylation are mediated by p300 and SIRT1, respectively. We found that PXR is directly acetylated by p300 at K109 by LC-MS/MS analysis. The K109Q acetylation mimicking mutant displayed reduced transcriptional activity and reduced ability to induce <i>cyp3A4</i> target gene mRNA and protein compared to the WT and the K109R acetylation-defective mutant. The diminished activity of the K109Q mutant appears to be due to impaired heterodimerization with RXRa and impaired binding of the PXR-RXRa heterodimer to DNA response elements. Furthermore, PXR acetylation appears to have an effect at the phenotypic level, in that pharmacological modulation of PXR acetylation levels can modulate its lipogenic function in mouse primary hepatocytes independent of a ligand. Moreover, the K109Q mutant displays impaired chemoprotective function based on morphological assessment of cells overexpressing K109Q and challenged with indomethacin, suggesting that K109 acetylation downregulates PXR's chemoprotective and perhaps anti-apoptotic functions, although this must be explored further. Notably, the K109R mutant displayed the WT phenotype, further supporting that acetylation itself, not just any arbitrary mutation confers the effect. Altogether, the data suggests that acetylation at K109 represents an overall "loss of function" effect on PXR activity. Implications of our findings are discussed in the context of known roles for PXR in transcription, health, and disease.</p>