Biochemistry Commons^™

Regulation Of Cytochrome C Functions By Phosphorylation, Gargi Mahapatra

Wayne State University Dissertations

The long term goal of my thesis research is to understand how tissue-specific

phosphorylations on the small mitochondrial protein, cytochrome c (Cytc), regulate its

functions, under both physiologically healthy and stressed conditions, and to identify the

cell signaling pathways targeting Cytc. Cytc is a functionally diverse protein that carries

electrons in the electron transport chain and plays a critical role in cellular apoptosis, two

diverse pathways that maintain cellular health that are active under diverse conditions.

Since Cytc plays a pivotal role in both these highly divergent pathways, regulation of the

protein is very important—phosphorylation of the protein under physiological …

Studies Towards Broadening The Substrate Profile And Regulation Of Histone Deacetylase 1, Dhanusha Ashanthi Nalawansha

Wayne State University Dissertations

Aberrant expression of histone deacetylase 1 (HDAC1) is implicated in multiple diseases, including cancer. As a consequence, HDAC1 has emerged as an important therapeutic target for drug development. HDAC1 regulates key cellular processes, such as cell proliferation, apoptosis, and cell survival, by deacetylating both histone and non-histone substrates. Due to the lack of simple tools to identify physiological substrates of HDAC1, the full spectrum of HDAC1 activities in the cell remains unclear. Here, we employed a substrate trapping strategy to identify cellular substrates of HDAC1. Using this approach, we identified mitosis-related protein Eg5 as a substrate. HDAC1 colocalizes with Eg5 …

The Unusual Substrate Specificity Of A Virulence Associated Serine Hydrolase From The Highly Toxic Bacterium, Francisella Tularensis, Alexander M. Farberg, Whitney K. Hart, R. Jeremy Johnson

Scholarship and Professional Work - LAS

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from …

Thermodynamics And Kinetics Of The Three-Way Junction Of Phi29 Motor Prna And Its Assembly Into Nanoparticles For Therapeutic Delivery To Prostate Cancer, Daniel W. Binzel

Theses and Dissertations--Pharmacy

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble in solution without the presence of metal ions. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Here the assembly mechanism, thermodynamic and enzymatic stabilities, and kinetics are examined in order to understand the stability behind this unique motif. Thermodynamic and kinetics studies found that the pRNA 3WJ formed out of …

Investigating And Expanding The Functionality Of Rna Catalysts: Studies Of The Hepatitis Delta Virus, The Hammerhead, And The Aminoacyl-Trna Synthetase-Like Ribozymes, Ji Chen

Open Access Dissertations

Ribozymes, just like protein enzymes, catalyze diverse chemical reactions. The first goal of this dissertation is to understand the mechanism of ribozyme-mediated phosphodiester cleavage reaction. Biochemical assays and X-ray crystallography were used for probing the active site of two small self-cleaving ribozymes, the hepatitis delta virus (HDV) ribozyme and the hammerhead ribozyme. Results presented here suggest that divalent metal ions play critical roles in the catalytic mechanisms of both the HDV and the hammerhead ribozymes. In the HDV ribozyme, the result is consistent with an active site Mg2+ being directly involved in catalysis. In the hammerhead ribozyme, however, Mg2+ ions …

Activation Of Erk And Nf-Κb During Hare-Mediated Heparin Uptake Require Only One Of The Four Endocytic Motifs, Madhu S. Pandey, Colton M. Miller, Edward N. Harris, Paul H. Weigel

Department of Biochemistry: Faculty Publications

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hepmediated signaling. Deletion of M1, M2 or …

Systems Perturbation Analysis Of A Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates For Cancer Therapeutics, Bhanwar Lal Puniya, Laura Allen, Colleen Hochfelde, Mahbubul Majumder, Tomáš Helikar

Department of Biochemistry: Faculty Publications

Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model's components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on …

Cadmium And Secondary Structure-Dependent Function Of A Degron In The Pca1p Cadmium Exporter, Nathan Smith, Wenzhong Wel, Miaoyun Zhao, Xiaojuan Quin, Javier Seravalli, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250–350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the …

Chloroplast Membrane Remodeling During Freezing Stress Is Accompanied By Cytoplasmic Acidification Activating Sensitive To Freezing 2, Allison C. Barnes, Christoph Benning, Rebecca Roston

Department of Biochemistry: Faculty Publications

Low temperature is a seasonal abiotic stress which restricts native plant ranges and crop distributions. Two types of low temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, SENSITIVE TO FREEZING 2 (SFR2) was identified as a protein which responds in a non-transcriptional manner to freezing. Here, we investigate the cellular conditions which allow SFR2 activation. Using a combination of isolated organelle, whole tissue and whole plant assays, we provide evidence that SFR2 is activated by changes …

Oma1 Links Mitochondrial Protein Quality Control And Tor Signaling To Modulate Physiological Plasticity And Cellular Stress Responses, Iryna Bohovych, Stavroula Kastora, Sara Christianson, Danelle Topil, Heejeong Kim, Teresa Fangman, You J. Zhou, Antoni Barrientos, Jaekwon Lee, Alistair J.P. Brown, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

A network of conserved proteases known as the intramitochondrial quality control (IMQC) system is central to mitochondrial protein homeostasis and cellular health. IMQC proteases also appear to participate in establishment of signaling cues for mitochondrion- to-nucleus communication. However, little is known about this process. Here, we show that in Saccharomyces cerevisiae, inactivation of the membrane-bound IMQC protease Oma1 interferes with oxidative-stress responses through enhanced production of reactive oxygen species (ROS) during logarithmic growth and reduced stress signaling via the TORC1-Rim15- Msn2/Msn4 axis. Pharmacological or genetic prevention of ROS accumulation in Oma1-deficient cells restores this defective TOR signaling. Additionally, inactivation …

Regulation Of Dj-1 By Glutaredoxin 1 In Vivo – Implications For Parkinson’S Disease, William M. Johnson, Marcin Golczak, Kyonghwan Choe, Pierce L. Curran, Olga Gorelenkova Miller, Chen Yao, Wenzhang Wang, Jiusheng Lin, Nicole M. Milkovic, Ajit Ray, Vijayalakshmi Ravindranath, Xiongwei Zhu, Mark A. Wilson, Amy L. Wilson-Delfosse, Shu G. Chen, John J. Mieyal

Department of Biochemistry: Faculty Publications

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH; and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has …

Inhibiting Hexamer Disassembly Of Human Udp-Glucose Dehydrogenase By Photoactivated Amino Acid Crosslinking, George Grady, Ashley Thelen, Jaleen Albers, Tong Ju, Jiantao Guo, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD⁺-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength …

Tumor Suppressive Role Of Sestrin2 During Colitis And Colon Carcinogenesis, Seung-Hyun Ro, Xiang Xue, Sadeesh K. Ramakrishnan, Chun-Seok Cho, Sim Namkoong, Insook Jang, Ian A. Semple, Allison Ho, Hwan-Woo Park, Yatrik M. Shah, Jun Hee Lee

Department of Biochemistry: Faculty Publications

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery …

Antisense Oligonucleotides: Treatment Strategies And Cellular Internalization, Colton M. Miller, Edward N. Harris

Department of Biochemistry: Faculty Publications

The clinical application of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific …

Polyglycine Acts As A Rejection Signal For Protein Transport At The Chloroplast Envelope, Joshua K. Endow, Agostinho Gomes Rocha, Amy J. Baldwin, Rebecca L. Roston, Toshio Yamaguchi, Hironari Kamikubo, Kentaro Inoue

Department of Biochemistry: Faculty Publications

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion …

Mutation In Atg5 Reduces Autophagy And Leads To Ataxia With Developmental Delay, Myungjin Kim, Erin Sandford, Damian Gatica, Yu Qiu, Xu Liu, Yumei Zheng, Brenda A. Schulman, Jishu Xu, Ian Semple, Seung-Hyun Ro, Boyoung Kim, R. Nehir Mavioglu, Aslihan Tolun, Andras Jipa, Szabolcs Takats, Manuela Karpati, Jun Z. Li, Zuhal Yapici, Gabor Juhasz, Jun Hee Lee, Daniel J. Klionsky, Margit Burmeister

Department of Biochemistry: Faculty Publications

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects’ cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast …

Novel Acyltranserases And Methods Of Using, Edgar B. Cahoon, Umidjon Iskandarov, Hae Jin Kim, Jillian Collins-Silva

Department of Biochemistry: Faculty Publications

Provided herein are novel acyltransferases and methods of using Such novel acyltransferases in making medium-chain fatty acids.

Can Chilling Tolerance Of C4 Photosynthesis In Miscanthus Be Transferred To Sugarcane?, Katarzyna Glowacka, Aasifuddin Ahmed, Shailendra Sharma, Tom Abbott, Jack C. Comstock, Stephen P. Long, Erik J. Sacks

Department of Biochemistry: Faculty Publications

The goal of this study was to investigate whether chilling tolerance of C₄ photosynthesis in Miscanthus can be transferred to sugarcane by hybridization. Net leaf CO₂ uptake (A_sat) and the maximum operating efficiency of photosystem II (Ф_PSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 day in controlled environment chambers. Two of three hybrids (miscanes), ‘US 84-1058’ and ‘US 87-1019’, did not differ significantly from the chilling tolerant M. xgiganteus ‘Illinois’ (Mxg), for A_sat, and Φ …

High-Throughput Mutation, Selection, And Phenotype Screening Of Mutant Methanogenic Archaea, Mary E. Walter, Alicia Ortiz, Casey Sondgeroth, Nathan M. Sindt, Nikolas Duszenko, Jennie L. Catlett, You Zhou, Shah R. Valloppilly, Christopher Anderson, Samodha Fernando, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or …

Pluralistic And Stochastic Gene Regulation: Examples, Models And Consistent Theory, Elisa N. Salas, Jiang Shu, Matyas F. Cserhati, Donald P. Weeks, Istvan Ladunga

Department of Biochemistry: Faculty Publications

We present a theory of pluralistic and stochastic gene regulation. To bridge the gap between empirical studies and mathematical models, we integrate pre-existing observations with our meta-analyses of the ENCODE ChIP-Seq experiments. Earlier evidence includes fluctuations in levels, location, activity, and binding of transcription factors, variable DNA motifs, and bursts in gene expression. Stochastic regulation is also indicated by frequently subdued effects of knockout mutants of regulators, their evolutionary losses/gains and massive rewiring of regulatory sites. We report wide-spread pluralistic regulation in ≈800 000 tightly co-expressed pairs of diverse human genes. Typically, half of ≈50 observed regulators bind to both …