Biochemistry Commons^™

Regulation Of Liver Mitochondrial Metabolism During Hibernation By Post-Translational Modification, Katherine E. Mathers

Electronic Thesis and Dissertation Repository

Hibernation, characterized by a seasonal reduction in metabolism and body temperature, allows animals to conserve energy when environmental conditions (e.g. temperature, food availability) are unfavourable. During hibernation, small mammals such as the 13-lined ground squirrel (Ictidomys tridecemlineatus) cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5 °C, and interbout euthermia (IBE), where metabolic rate and body temperature rapidly increase and are maintained at euthermic levels several hours. Suppression of metabolism during entrance into torpor is paralleled by rapid suppression of liver mitochondrial metabolism. In my thesis, I …

Role Of Asparagine As A Nitrogen Signal And Characterization Of A Nitrogen Responsive Glutamine Amidotransferase, Gat1_2.1 In Arabidopsis Thaliana, Shrikaar Kambhampati

Electronic Thesis and Dissertation Repository

Maintaining the proper balance between carbon (C) and nitrogen (N) metabolism is critical to the sustained growth of organisms. In plant leaves, this balance is achieved by photoperiod dependent cross-talk between the processes of photosynthesis, respiration, and amino acid metabolism. A crucial mechanism in maintaining C/N balance is the GS/GOGAT cycle, which is well known to serve as a cross-road between C and N metabolism. Importantly, non-photosynthetic tissues (e.g. roots, germinating seeds) lack a sufficient supply of carbon skeletons under high N conditions and hence may resort to other mechanisms, along with the GS/GOGAT cycle, to achieve proper C/N balance. …

Nbpmf: Novel Network-Based Inference Methods For Peptide Mass Fingerprinting, Zhewei Liang

Electronic Thesis and Dissertation Repository

Proteins are large, complex molecules that perform a vast array of functions in every living cell. A proteome is a set of proteins produced in an organism, and proteomics is the large-scale study of proteomes. Several high-throughput technologies have been developed in proteomics, where the most commonly applied are mass spectrometry (MS) based approaches. MS is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification, quantification, and post translational modification (PTM) characterization in proteomics research. There are usually two different ways to identify proteins: top-down and bottom-up. Top-down approaches …

Identification Of Novel Binding Partners And Substrates Of Histone H3k4 Specific Demethylase Kdm5b/Jarid1b, Qi Fang

Electronic Thesis and Dissertation Repository

It has been 50 years since the first histone methylation was identified. From then, numerous studies have verified that histone methylation neutralizes the positive charges of lysine, alters chromatin architectures, and recruits other DNA binding complexes. In this study, we examined the molecular basis of a histone demethylase – KDM5B in both its substrate and binding machineries. We hypothesized that KDM5B has alternative binding partners and substrates besides its known target H3K4me3. By utilizing biochemical approaches such as in vitro pull-down assays and fluorescence polarization assays, we were able to identify potential KDM5B binding partners – H2BK43me0/2. Also, we reinvestigated …

An Investigation Of The Ck2-Dependent Phosphoproteome Using Inhibitor Refractory Ck2-Alpha, Edward Cruise

Electronic Thesis and Dissertation Repository

Protein kinase CK2 is a constitutively active serine/threonine kinase that is overexpressed in several human cancers, and by virtue of the vast number of putative substrates in the phosphoproteome, is implicated in the regulation of numerous cellular processes. Consequently, CK2 is an emerging therapeutic target with many CK2 inhibitors having been developed. An example of one such inhibitor is the clinical stage compound CX-4945. Although highly selective for CK2, the ATP competitive CX-4945 has demonstrated affinity for other kinases. Unique features of the catalytic pocket of CK2 have allowed for the development of inhibitor refractory mutants, which have since been …

Regulation Of C-Raf Stability By The Ranbpm/Ctlh Complex, Christina J. Mctavish

Electronic Thesis and Dissertation Repository

RanBPM is an evolutionarily conserved multi-domain protein that has been implicated in the regulation of several cellular process, including protein stability, cell migration, gene transcription, and apoptosis. RanBPM is identified as a key member of the CTLH complex, an orthologous complex to a yeast E3 ubiquitin ligase complex, the exact function of which remains unknown. Previously, our laboratory identified RanBPM as an inhibitor of the ERK1/2 pathway through the modulation of C-RAF protein levels. This study shows that RanBPM-mediated degradation of C-RAF occurs through the proteasome and the entire CRA domain of RanBPM is necessary for direct interaction with C-RAF …

Deciphering Sulfur Amino Acid Metabolism In Developing Seeds Of Common Bean, Jaya Joshi

Electronic Thesis and Dissertation Repository

With increasing food insecurity in the populated world, the number of people affected by chronic undernourishment is also increasing. Alone, protein energy malnutrition is linked to 6 million deaths annually. Despite being a good source of protein and dietary fibre, the quality of bean protein is limited because of sub optimal levels of essential sulfur amino acids: methionine and cysteine. Levels of cysteine and methionine in developing seeds have an inverse relationship with the non-protein sulfur amino acid S-methyl-cysteine (S-methylCys) and dipeptide g-glutamyl-S-methyl-cysteine (g-Glu-S-methylCys).

One of the strategies to improve protein quality in …

Structural And Functional Characterization Of Non-Homologous End Joining Factors, Huasheng Wang

Electronic Thesis and Dissertation Repository

DNA double strand breaks represent the most toxic form of DNA damage. In mammals, non-homologous end-joining (NHEJ) is the primary DNA repair pathway for such damage, preventing both carcinogenesis and accelerated aging. Structural understanding of this repair pathway has received considerable attention, but has been significantly limited by the inability to obtain structures of higher order nucleoprotein complexes. A main obstacle in this respect has been difficulty in obtaining highly purified proteins, sufficient for structural determination. Improved protein expression and purification methods developed in this thesis permitted several NHEJ complexes to be selected for structural studies. Among these, Ku70-DNA and …

Expression, Characterization And Metallation Studies Of Human Metallothionein Isoform 2a Using Electrospray Ionization Mass Spectrometry, Devika P. Jayawardena

Electronic Thesis and Dissertation Repository

Maintenance of the homeostasis of zinc (Zn) is very important in regulating bodily functions. There are over 300 Zn dependent enzymes identified, where Zn plays a structural or catalytic role. However, excess of Zn in a cell is toxic and free Zn ions are tightly controlled. Metallothioneins (MTs) are small cysteine rich proteins, which can bind up to seven Zn ions and act as a Zn reservoir. The MT2a isoform is predominantly found in the liver. My research focused on the overexpression of human MT2a in Escherichia coli and the investigation of Zn binding pathways of MT2a in vitro. …

Investigation Of The Interactions Between Elg1 And Pcna Involved In The Dna Damage Response, Jingwei Yan

Electronic Thesis and Dissertation Repository

Elg1, the major component of an alternative PCNA loader, is vital in maintaining chromosomal stability. PCNA is an essential coordinator for DNA replication and the damage response through protein-protein interactions. The N-terminus of Elg1 interacts with PCNA and preferentially SUMOylated PCNA (sPCNA) and contains five putative motifs for PCNA and SUMO binding. How these motifs contribute to PCNA interactions is unclear. In this study, we biochemically characterized the interactions between Elg1 and PCNA and found that i) two PCNA-interacting protein boxes (PIP boxes) participate in the interaction with PCNA, which may be interfered by the SUMO-interacting motifs (SIMs); ii) the …

Characterizing The Cofactor Specificity Of Nqo2, Shahed Al Massri

Electronic Thesis and Dissertation Repository

Quinone Reductase 2 (NQO2) is a part of an enzyme family implicated in detoxification of the cell. This enzyme differs from its highly similar sister protein Quinone Reductase 1 (NQO1) by its unique cofactor preference for dihydro-nicotinamide riboside (NRH) instead of NADH. Cellular levels of NRH have not been characterized, contributing to the mystery surrounding the physiological role of NQO2. This project focused on identifying what determines the cofactor specificity of NQO2, and engineering it to use NADH instead of NRH. The investigation included optimization of protein purification, an in silico analysis that revealed the different stereochemistry of NQO2/1 from …

Metalation And Structural Properties Of Apo-Metallothioneins, Gordon W. Irvine

Electronic Thesis and Dissertation Repository

Metals are required by a quarter of all proteins to achieve their biological function, whether in an active site involved in catalytic chemistry or in a structural capacity. Metals are tightly regulated at the cellular level due to their propensity to cause unwanted side reactions and to be scavenged for use by pathogens. One of the proteins involved in this regulation of metal homeostasis is metallothionein (MT) which is a small, cysteine rich protein primarily involved in the regulation of zinc and copper homeostasis and heavy metal detoxification. MT is unique in its high cysteine content (~30% of the residues), …

Investigating E2f Independent Cell Cycle Control And Tumor Suppression By Prb, Michael J. Thwaites

Electronic Thesis and Dissertation Repository

Cellular division is primarily controlled at the G1 to S-phase transition of the cell cycle by the retinoblastoma tumor-suppressor protein (pRB). The ability of pRB to restrict S-phase entry is primarily attributed to the repression of E2F transcription factors required to upregulate cell cycle target genes necessary for cellular division. Interestingly, while pRB is disrupted in the vast majority of human cancers, mutations typically target upstream regulators of pRB leading to inactivation through hyperphosphorylation. The rarity of direct pRB mutations suggests that the regulation of the cell cycle by pRB may involve additional mechanisms outside of E2F repression, as this …

Assessing The Role Of Ku70 Vwa Domain Phosphorylation In The Inhibition Of Aurora B And Activation Of The Dna Damage Response, Elizabeth A. Walden

Electronic Thesis and Dissertation Repository

Ku is a key component of the Non-Homologous End Joining DNA repair pathway. Recently, a function for Ku in DNA damage response (DDR) signalling was identified through studies exploring a Ku70 S155D phosphomimetic mutant. We hypothesize that Ku70 S155D mimics phosphorylation of Ku70 in response to DNA damage, and that Ku S155 phosphorylation inhibits Aurora B and causes sustained DDR activation. In this study we show that the S155D mutant is competent for heterodimerization, and its expression does not induce DNA damage. Phosphorylated Ku70 associates with Aurora B by co-immunoprecipitation and this association was demonstrated in situ with Ku70 S155D. …

Quantitative Proteomic Characterization Of Cx-4945, A Clinical Stage Inhibitor Of Protein Kinase Ck2, Adam J. Rabalski

Electronic Thesis and Dissertation Repository

Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to …

Dual-Active Genome-Editing Reagents, Jason M. Wolfs

Electronic Thesis and Dissertation Repository

Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived …