Immunosuppressants (such as azathioprine and 6‐mercaptopurine) are widely used in the management of patients having rheumatic diseases, inflammatory bowel diseases, hematological malignancies and organ transplant rejections. However, the adverse effects and effectiveness of these drugs are dependent on the metabolism by the enzyme, thiopurine S‐methyltransferase (TPMT), inside the body and the activity of this enzyme is determined by its genetic polymorphism. This study mainly focused on four known mutations, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C which were detected by three sets of primers, G238C, G460A and A719G targeting exons 5, 7 & 10 of the TPMT gene. Patient blood was collected from patients who had a clinical need of knowing the TPMT level (n=202). The TPMT phenotypic status of patient was determined by measuring the enzyme activity of red blood cell lysates by Enzyme‐Linked Immunosorbent Assay (ELISA) commercially available. On the other hand, the genotype was reflected by the sequencing results generated after DNA extraction from whole blood, followed by amplification, purification and DNA sequencing by the targeted primers. The majority of patients (92%) showed normal to high TPMT enzyme activity level (>17 U) and the remaining 8% was under the category of borderline activity (between 7 U to 17 U). None of them had low or deficient activity. The mean TPMT enzyme activity of all samples was 22.9 U ranging from 7.8 U to 54.1 U. No observable difference was found between male and female. The largest group of patients was having rheumatic diseases, with enzyme activity levels from 7.8 U to 54.1 U (mean of 22.8 U) which was very close to the overall findings. Also, there was no direct relationship between the lowest white blood cell count and the TPMT activity of each patient. Low white blood cell counts were not usually associated with lower TPMT enzyme activity. From the DNA sequencing results, 62.5% of the samples (n=104) had no genetic abnormalities found, 31.7% were found to have a heterozygous allele C/T and G/A at position 474 which was known to be a silent mutation with no amino acid alteration and hence was not functionally defective. Only 4.8% had heterozygous allele A/G and T/C at position 719 and one sample was found to have heterozygous allele at both positions 719 and 474. There was no significant difference in the TPMT enzymatic activity between the samples with genetic abnormalities and those without genetic abnormalities (means of TPMT enzymatic activity were 17.3 U and 21.5 U respectively, p=0.13). And also, no apparent correlation was found among the TPMT enzymatic levels, the genetic abnormalities and the disease groups. In conclusion, the individual differences in the TPMT enzyme activity were resulted from the allelic variation at the TPMT locus, it was important to fully understand the allelic variation at the TPMT gene locus. The ghenotypic analysis could be extended to the detection of all the ten exons including their spice‐site junctions and 5’ flanking promoter region of the TPMT gene by PCR single strand conformation polymorphism in future studies. / published_or_final_version / Pathology / Master / Master of Medical Sciences

This is the first study to explore the ability of an enzyme to recognize and repair spontaneous age-dependent damage to its own sequence. Protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase (PCM) is known to repair damage that arises from a spontaneous isomerization of aspartyl and asparaginyl residues in other proteins during aging. As PCM contains several conserved aspartyl and asparaginyl residues, this dissertation tested whether PCM can serve as a methyl acceptor in its own methylation reaction. In investigating the ability of PCM to automethylate, it was discovered that PCM is damaged. The mechanism of this automethylation reaction was determined to be an intermolecular, high affinity, slow turnover reaction and was limited to a subpopulation of damaged PCM molecules, termed ��PCM. / Graduation date: 1996

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Medicinal Chemistry. Title from title-page of electronic thesis. Bibliography: leaves 103-114.

Thesis (Ph. D.)--Cornell University, April, 2008. / Vita. Includes bibliographical references (leaves 151-166).

Thesis (Ph. D.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 165-196).

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the stepwise transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) to the amino headgroup of PE. Successive methylation results in the formation of the two intermediates, phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N, N-dimethylethanolamine (PDME), and the final product phosphatidylcholine (PC). PE N-methyltransferase is an integral membrane protein localized primarily in the endoplasmic reticulum (microsomal fraction) of liver. PE-, PMME- and PDME-dependent PE N-methyltransferase activities were purified from Triton X-100 solubilized microsomes 429-, 1542- and 832-fold, respectively. The purified enzyme was composed of a single 18.3 kDal protein as determined by SDS-PAGE. Molecular mass analysis of purified PE N-methyltransferase (in Triton X-100 micelles) by gel filtration on Sephacryl S-300 indicated the enzyme existed as a 24.7 kDal monomer. PE N-methyltransferase catalyzed the complete conversion of PE to PC and had a pH optimum of 10 for all three steps. A Triton X-100 mixed micelle assay was developed to assay PE-, PMME- and PDME-dependent activities of both pure and microsomal PE N-methyltransferase. The AT-terminal amino acid sequence of rat liver PE N-methyltransferase and the recently cloned 23.1 kDal S. cerevisiae PEM 2 were found to be 35% homologous. Double reciprocal plots for PE N-methyltransferase at fixed Triton X-100 concentrations and increasing PE, PMME or PDME were highly cooperative. Similar cooperative effects were noted when phospholipid was fixed and Triton X-100 increased. The cooperativity could be partially abolished if a fixed mol% of nonsubstrate phospholipid such as PC was included in the assay. This would indicate that PE N-methyltransferase has specific binding requirements for a site(s) in contact with the micellar substrate. The occupation of this boundary layer by phospholipid is essential for full expression of enzyme activity. Kinetic analysis revealed that PMME and PDME methylation followed an ordered Bi-Bi mechanism. The overall mechanism involves initial binding of PE to a common site and successive methylation steps involving the binding and release of AdoMet and S-adenosyl-L-homocysteine, respectively. Cysteine residue(s) (which are rapidly oxidized in the absence of reduced thiols) are involved in the catalytic mechanism. Reverse-phase HPLC was used to fractionate the phospholipid products of PE N-methyltransferase into individual molecular species. Substrate specificity experiments on PE N-methyltransferase in vitro and in vivo revealed no selectivity for any molecular species of diacyl PE, PMME or PDME. The PE-derived PC, which is rich in 16:0-22:6, is rapidly remodeled to conform to the molecular species compositon of total hepatocyte PC in vivo . The 18.3 kDal PE N-methyltransferase was found to be a substrate for cAMP-dependent protein kinase in vitro. However, only 0.25 mol phosphorus/mol of PE Af-methyltransferase was incorporated, with no observed effect on activity. Studies on PE N-methyltransferase regulation in choline-deficient rat liver indicated that activity changes were due to elevated levels of cellular PE. Immunoblotting of choline-deficient liver microsomes or hepatocyte membranes with a anti-PE N-methyltransferase antibody revealed no alteration in enzyme mass. While more work is needed, initial indications are that hepatic PE N-methyltransferase is a constitutive enzyme regulated primarily by substrate and product levels. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

<div><br></div><div>Plants are constantly exposed to biotic and abiotic factors throughout their developmental stages which threaten their growth and productivity. Environmental stresses limit crop productivity and are likely to increase in severity due to the drastic and rapid changes in global climate. In this project, we studied the genetic factors that contribute to plant adaption to pathogens and other environmental factors in tomato. The results of these are presented in chapters 2-4 of this thesis. Chapter 1 covers background information and the review of the current literature in plant responses to biotic and abiotic stress. Chapter 2 deals with functional analysis of tomato histone methyltransferases SDG33 and SDG34 and their role in plant defense and stress tolerance. Chapter 3 focuses on the role of SDG33 and SDG34 on plant responses to Nitrogen. Finally, Chapter 4 summarizes the results from a reverse genetic screen using CRISPR cas9 genome editing to identify Receptor Like Cytoplasmic Kinases (RLCKs) required for plant resistance to fungal pathogens. </div><div>Plant responses to environmental cues are underpinned by rapid and extensive transcriptional reprogramming. Post translational modification of histones orchestrate these reprogramming and cellular responses by altering chromatin structure and establishing permissive or repressive states. Histone lysine methylation (HLM) is a principal modification of chromatin that affects various cellular processes. HLM is mediated by histone methyltransferases (HMTs) that deposit methyl groups to specific lysine residues on n-terminal histones tails. Although it is known that chromatin modifications occur in response to environmental cues, the mechanisms by which this is achieved, and the biological functions of HMTs are poorly understood. The function of tomato histone methyltransferases Set Domain Group (SDG)33 and SDG34 in biotic and abiotic stress responses were studied using tomato mutants generated through CRISPR/cas9 genome editing. </div><div>SDG33 and SDG34 genes were induced by pathogens, drought stress, the plant hormones methyl jasmonate, salicylate and abscisic acid. The sdg33 and sdg34 mutants display altered global HLMs. SDG34 is required for global H3K36 and H3K4 mono, di- and tri-methylation while SDG33 is primarily responsible for di- and tri- H3K36 and H3K4 methylation. Tomato SDG33 and SDG34 are orthologues of the Arabidopsis SDG8, an H3K4 and H3K36 methyl transferase previously implicated in plant immunity and plant growth through epigenetic control of Carotenoid Isomerase (CCR2) and other target genes. However, the tomato sdg33 or sdg34 single mutants showed no altered responses to fungal and bacterial pathogens likely due to functional redundancy of the tomato SDG33 and SDG34 genes consistent with their overlapping biochemical activities. Interestingly, tomato SDG33 or SDG34 genes rescued the disease susceptibility and early flowering phenotypes of Arabidopsis sdg8 mutant. Expression of CCR2 gene is completely inhibited in Arabidopsis sdg8 mutant attributed to loss of H3K36 di- and tri methylation at CCR2 chromatin. CCR2 gene expression was partially restored by transgenic expression of tomato SDG33 or SDG34 genes in Arabidopsis sdg8. In tomato, the single CCR2 gene is expressed independent of SDG33 or SDG33 genes suggesting that the genomic targets of the tomato HMTs are different. Unexpectedly, sdg33 and sdg34 plants were more tolerant to osmotic stress, maintain a higher water status during drought which translated to better survival after drought. Tolerance of sdg33 and sdg34 to drought stress is accompanied by higher expression of drought responsive genes. Collectively, our data demonstrate the critical role of tomato HLM in pathogen and stress tolerance likely through the regulation of gene expression.</div><div>In parallel, we characterized the role of SDGs in mediating nitrogen responses in tomato. The results are described in Chapter 2. Few studies have focused on the role of histone lysine methylation in regulating changes to nutrient availability. Transcriptome analysis in the shoot and roots showed that SDG33 and SDG34 have both overlapping and distinct regulated targets in tomato. In response to nitrogen, 509 and 245 genes are regulated by both SDG33 and SDG34 in response to nitrogen states in the roots and shoot respectively. In the roots these genes were enriched with GO terms such as ‘regulation of gene expression’, regulation of N metabolism’ and ‘regulation of hormone stimuli’. ‘Response to stimulus’, ‘photosynthesis’ and ‘N assimilation’ were the biological processes significantly enriched in the shoots. Overall, we show that SDG33 and SDG34 are involved in regulating nitrogen responsive gene expression and hence physiological nitrogen responses in the roots and shoots. </div><div>We also studied the Set Domain Group 20 (SlSDG20) an orthologue of Arabidopsis SDG25 in tomato. The details of our observations are presented in Chapter 3. SlSDG20 belongs to class III HMTs, it has the SET, Post-SET domain and GYF domain important for proline-rich sequence recognition. SlSDG20 is highly induced by B. cinerea, Methyl Jasmonate and Ethylene. To further understand the functions of SlSDG20 in tomato physiological development and plant immunity we generated slsdg20 knockout mutants through CRSIPR/Cas9. We identified one homozygous slsdg20 mutant with 151bp deletion in an exon immediately before the SET domain. Global methylation assay on the slsdg20 mutant confirmed that SlSDG20 is an H3K4 methyltransferase. The slsdg20 mutant is shorter than the wild type, produce more adventitious shoots causing prolific branching, and produce narrow leaves. Further, the mutant produces abnormal fruit and few seeds that hardly germinate. The slsdg20 mutant is highly susceptible to B. cinerea compared to the wild type. In response to Pst DC3000, slsdg20 mutant plants are comparable of the wild type. Resistance to hrcC strain of Pst DC3000 was impaired in the slsdg20 mutant, suggesting a possible role of SlSDG20 in PTI. In sum, tomato SDG20 is regulates plant immunity and plant growth including fertility.</div><div>The final chapter focuses on tomato Receptor like cytoplasmic kinases (RLCKs). Plants perceive the presence of pathogens through Pattern Recognition Receptors (PRR) which are predominantly RLKs, and subsequently recruit RLCKs to signal to downstream regulators of defense responses. Many RLCKs were characterized from Arabidopsis for their role in signalling of responses to bacterial infection. An example of RLCKs is Arabidopsis BIK1 which is implicated in signal transmission of pathogen recognition event at the cell surface. The tomato genome encodes 647 RLK/RLCKs comprising about 2% of its predicted genes. The functions of most of these predicted tomato RLCKs and RLKs have not been determined. Previously, our lab characterized the Arabidopsis BIK1 and tomato TPK1b RLCKs for fungal resistance. Here, we conducted a reverse genetic screen focused on BIK1 and TPK1b related tomato RLCKs to identify a subset with defense functions. Virus induced gene silencing and pathogen assays conducted on 15 RLCKs identified four RLCK genes with potential role in plant immunity. Then, tomato knock out mutants were generated for four RLCK genes through CRISPR/cas9 genome editing to validate the VIGS data. Subsequently, we demonstrated that TPK07, TPK09, TPK011 and TRK04 are required for resistance to B. cinerea. The data are supported by the pathogen induced expression of these genes. Furthermore, trk04 seedlings are impaired in seedling growth responses to Jasmonic acid. Our study establishes that tomato TPK07, TPK09, TPK011 and TRK04 contribute to defense against B. cinerea but their mechanism of function needs to be elucidated in future studies</div><div><br></div>

<p>CaaX proteins are involved in many key cellular processes such as proliferation, differentiation, trafficking, and gene expression. CaaX proteins have four specific C-terminal amino acids designated as a CaaX motif, where the “C” is a cysteine, “a” are aliphatic residues, and “X” represents one of several amino acids. Proteins with this motif undergo three post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the –aaX residues and methylation of the isoprenylated cysteine, which is necessary for their localization in the cell and function. Due to involvement of CaaX proteins in many critical signaling pathways, mutations in CaaX proteins can result in a wide variety of disorders and carcinomas. Most notably, mutants in the <i>KRAS</i> gene are associated with 90% of pancreatic cancers and 30% of all cancers. Isoprenylcysteine carboxyl methyltransferase (Icmt), an integral membrane protein in the endoplasmic reticulum, is the only known protein responsible for the post-translational α-carboxyl methylesterification of the C-terminus of CaaX proteins. Cells with Icmt deficiency causes the small G-protein, K-ras, to be mislocalized and decreases downstream signaling of K-ras. Thus, our goal is to better understand the structure and methylation mechanism of Icmt in order to inhibit mutant K-ras in oncogenic cells and aid in the creation of a chemotherapeutic for pancreatic cancer. </p> <p>Icmt studies have focused on the founding member of the Icmt family, Ste14. Ste14 is expressed in <i>Saccharomyces cerevisiae </i>(<i>S. cerevisiae</i>)<i> </i>and shares high homology with the human Icmt (hIcmt), which has yet to be functionally purified. Specifically, hIcmt and Ste14 share 63% similarity and 41% identity, mostly within the C-termini of the proteins. First, we optimized expression and purification of Ste14 in order to generate a larger yield of protein, which is necessary for many biophysical techniques. Infection of Sf9 cells with a baculovirus expressing an N-terminally 10-His-tagged and 3-myc-tagged Ste14 (His-Ste14), increased protein expression between four and five-fold compared to our yeast model and used significantly less starting materials. We also performed a detergent screen for the purification of His-Ste14 from insect cell expression. We concluded that <i>n</i>-Dodecyl-β-D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), and heptaethylene glycol monododecyl ether (C₁₂E₇) were detergents that stabilize His-Ste14 for further biophysical techniques. Additionally, we found 1xEQ buffer at pH 6.0 resulted in the most homogenous His-Ste14 sample.</p> <p>Second, we sought to elucidate the SAM binding/ SAH release mechanism of His-Ste14 by utilizing a combinatorial method of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy analysis. We used SDSL-EPR to determine the conformational dynamics of His-Ste14 with and without SAM. EPR is an attractive method to study conformational changes of proteins as it is done in solution and requires relatively small amounts of protein. We generated a library of 46 non-conserved single cysteine mutants introduced into cysteine-less His-Ste14 (His-Ste14-TA). The cysteine residues engineered into His-Ste14-TA were in the cytosolic portion of the protein to ensure efficient labeling and were tested for methyltransferase activity levels. From crude membranes, only nineteen mutants retained activity levels of ≥50% of His-Ste14-TA, which were then purified and tested for methyltransferase activity levels. Eight purified mutants were selected as candidates for EPR with activity levels of ≥50% of His-Ste14-TA. Once optimized, we introduced a nitroxide spin label, 1-oxyl-2,2,5,5-tetrametylpyrroline-3-methyl)-methanethiosulfonate (MTSL), to several of the purified single cysteine mutants. Then, we evaluated protein dynamics during the methylation reaction by monitoring mobility of the MTSL-labelled residue upon addition of SAM. Overall, our structural and biochemical analyses will be used to ascertain the structural dynamics associated with SAM binding of this unique methyltransferase.</p> <p>Additionally, we were able to incorporate His-Ste14 in nanodiscs. Nanodiscs mimic the membrane of a cell and are a more native-like environment that detergent micelles or liposomes. Since nanodiscs are conducive to many biophysical techniques, unlike detergents, we have begun preliminary studies to better understand the structure of Ste14. Techniques we have begun to pursue are negative stain electron microscopy (EM), single particle cryo-electron microscopy (cryo-EM), and X-ray crystallography. </p> <p>Finally, we previously showed Ste14 functions as a dimer or higher order oligomer. Ste14 is comprised of six transmembrane (TM) domains in which TM1 contains a putative dimerization motif, G₃₁XXXG₃₅XXXG₃₉, where G is a glycine amino acid residue and X is a subset of hydrophobic amino acids. Using cysteine-scanning mutagenesis, we characterized TM1 cysteine mutants for their effects on protein expression, activity, and stability. We determined residues S27, Y28, L30, G31, G35, and G39 are critical for maintaining activity levels. Additionally, residues M25, T26, Y28, F41, P42, and Q43 were found to form strong dimers through the addition of sulfhydryl specific cross-linkers and immunoblot analysis. Recently, the purification of dimeric Ste14 from aggregated protein components via size exclusion chromatography (SEC) was improved for further experimentation. The purified, monodispersed, His-Ste14 underwent size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small-angle X-ray scattering (SAXS) to confirm the dimerization state of Ste14. Together, we have used many biochemical and biophysical methods to gain insight about the structure, function, and mechanism of Ste14. Ultimately, our studies will be utilized to design more potent therapeutics to minimize K-Ras signaling in cancer cells.</p>

No description available.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy