Other Biochemistry, Biophysics, and Structural Biology Commons^™

Stress Adaptation In A Pathogenic Fungus, Alistair J. P. Brown, Susan Budge, Despoina Kaloriti, Anna Tillmann, Mette D. Jacobsen, Zhikang Yin, Iuliana V. Ene, Iryna Bohovych, Doblin Sandai, Stavroula Kastora, Joanna Potrykus, Elizabeth R. Ballou, Delma S. Childers, Shahida Shahana, Michelle D. Leach

Department of Biochemistry: Faculty Publications

Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of …

Stress-Triggered Activation Of The Metalloprotease Oma1 Involves Its C-Terminal Region And Is Important For Mitochondrial Stress Protection In Yeast, Iryna Bohovych, Garrett Donaldson, Sara Christianson, Nataliya Zahayko, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Background: Oma1 is a conserved membrane-bound protease that forms a high molecular mass complex.

Results: Oma1 activity is induced by stress stimuli and required for survival. The activation is linked to changes in Oma1 oligomer stability and involves its C-terminal region.

Conclusion: Oma1 function is activated by mitochondrial stress and is important for stress tolerance.

Significance: Novel insights into Oma1 function and a potential stress activation mechanism are provided.

Proline Biosynthesis Is Required For Endoplasmic Reticulum Stress Tolerance In Saccharomyces Cerevisiae, Xinwen Liang, M. B. Dickman, Donald F. Becker

Department of Biochemistry: Faculty Publications

Background: Proline is an important amino acid for stress resistance in different organisms.

Results: Depletion of proline biosynthesis disrupts redox homeostasis and increases sensitivity to endoplasmic reticulum (ER) stress in yeast.

Conclusion: Proline biosynthesis is critical for maintaining the intracellular redox environment and the UPR during ER stress.

Significance: Proline metabolism is shown to have an important role in ER stress tolerance that was previously unknown.

Protein/Protein Interactions In The Mammalian Heme Degradation Pathway Heme Oxygenase-2, Cytochrome P450 Reductase, And Biliverdin Reductase, Andrea L. M. Spencer, Ireena Bagai, Donald F. Becker, Erik R. P. Zuiderweg, Stephen W. Ragsdale

Department of Biochemistry: Faculty Publications

Background: Heme oxygenase, cytochrome P450 reductase, and biliverdin reductase are the key enzymes in heme degradation.

Results: Specific electrostatic and hydrophobic interactions form the binding interface between heme oxygenase and cytochrome P450 reductase. Conclusion: Heme oxygenase binds cytochrome P450 reductase dynamically and biliverdin reductase very weakly.

Significance: Characterizing interactions among proteins involved in heme degradation are crucial to understanding heme homeostasis.

Plant Acyl-Coa:Lysophosphatidylcholine Acyltransferases (Lpcats) Have Different Specificities In Their Forward And Reverse Reactions, Ida Lager, Jenny Lindberg Yilmaz, Xue-Rong Zhou, Katarzyna Jasieniecka, Michael Kazachkov, Peng Wang, Jitao Zou, Randall Weselake, Mark A. Smith, Shen Bayon, John M. Dyer, Jay Shockey, Ernst Heinz, Allan Green, Antoni Banas, Sten Stymne

Department of Biochemistry: Faculty Publications

Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine.

Results: Plant LPCATs were expressed in yeast and biochemically characterized.

Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions.

Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid.

Emerging Regulatory Paradigms In Glutathione Metabolism, Yilin Liu, Annastasia S. Hyde, Melanie A. Simpson, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes …

Emerging Roles For Hyaluronidase In Cancer Metastasis And Therapy, Caitlin O. Mcatee, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past five years have clarified many apparent contradictions, (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature …

Mechanisms Underlying The Exquisite Sensitivity Of Candida Albicans To Combinatorial Cationic And Oxidative Stress That Enhances The Potent Fungicidal Activity Of Phagocytes, Despoina Kaloriti, Mette Jacobsen, Zhikang Yin, Miranda Patterson, Anna Tillmann, Deborah A. Smith, Emily Cook, Tao You, Melissa J. Grimm, Iryna Bohovych, Celso Grebogi, Brahm H. Segal, Neil A.R. Gow, Ken Haynes, Janet Quinn, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus

Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity

of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual

stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro.We also show that the high

fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as

their pharmacological attenuation with …

New Clox Systems For Rapid And Efficient Gene Disruption In Candida Albicans, Shahida Shahana, Delma S. Childers, Elizabeth R. Ballou, Iryna Bohovych, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via …

Saturated Free Fatty Acids Induce Cholangiocyte Lipoapoptosis, Sathish Kumar Natarajan, Sally A. Ingham, Ashley M. Mohr, Cody J. Wehrkamp, Anuttoma Ray, Sohini Roy, Sophie C. Cazanave, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation …

Xiap Antagonist Embelin Inhibited Proliferation Of Cholangiocarcinoma Cells, Cody J. Wehrkamp, Ashley R. Gutwein, Sathish Kumar Natarajan, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 …

Oxidative Stress, Redox Signaling, And Autophagy: Cell Death Versus Survival, Juliana Navarro-Yepes, Michaela Burns, Anandhan Annadurai, Oleh Khalimonchuk, Luz Maria Del Razo, Betzabet Quintanilla-Vega, Aglaia Pappa, Mihalis I. Panayiotidis, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Significance: The molecular machinery regulating autophagy has started becoming elucidated, and a number of studies have undertaken the task to determine the role of autophagy in cell fate determination within the context of human disease progression. Oxidative stress and redox signaling are also largely involved in the etiology of human diseases, where both survival and cell death signaling cascades have been reported to be modulated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent Advances: To date, there is a good understanding of the signaling events regulating autophagy, as well as the signaling processes by which alterations in …

Kinetic And Structural Characterization Of Tunnel-Perturbing Mutants In Bradyrhizobium Japonicum Proline Utilization A, Benjamin W. Arentson, Min Luo, Travis A. Pemberton, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ¹-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall …

Evidence That The C‑Terminal Domain Of A Type B Puta Protein Contributes To Aldehyde Dehydrogenase Activity And Substrate Channeling, Min Luo, Shelbi Christgen, Nikhilesh Sanyal, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress …

Homogeneous Low-Molecular-Weight Heparins With Reversible Anticoagulant Activity, Yongmei Xu, Chao Cai, Kasemsiri Chandarajoti, Po-Hung Hsieh, Lingyun Li, Truong Q. Pham, Erica M. Sparkenbaugh, Juzheng Sheng, Nigel S. Key, Rafal Pawlinski, Edward N. Harris, Robert J. Linhardt, Jian Liu

Department of Biochemistry: Faculty Publications

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Pharmacological Correction Of Obesity-Induced Autophagy Arrest Using Calcium Channel Blockers, Hwan-Woo Park, Haeli Park, Ian A. Semple, Insook Jang, Seung-Hyun Ro, Myungjin Kim, Victor A. Cazares, Edward L. Stuenkel, Jung-Jae Kim, Jeong Sig Kim, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome–lysosome fusion in liver, preventing accumulation of …

Hepatoprotective Role Of Sestrin2 Against Chronic Er Stress, Hwan-Woo Park, Haeli Park, Seung-Hyun Ro, Insook Jang, Ian A. Semple, David N. Kim, Myungjin Kim, Myeongjin Nam, Deqiang Zhang, Lei Yin, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAATenhancer- binding protein beta (c/EBPβ). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the …

Molecular Evolution Of Protein-Rna Mimicry As A Mechanism For Translational Control, Assaf Katz, Lindsey Solden, S. Betty Zou, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the …

Biochemical Assay Optimization And Computational Screening Efforts To Identify Potential Luxs Inhibitors, Keeshia Q. Wang

Master's Theses

Quorum sensing (QS) is a process of coordination of bacterial gene expression in response to cell population. System two QS is regulated by the small signaling molecule autoinducer-2 (AI-2) and is implicated in the infectious behaviors of various bacterial species. AI-2 is biosynthesized from S-ribosylhomocysteine (SRH) by the enzyme LuxS and induces interspecies cell-to-cell communication. Inhibition of LuxS would therefore inhibit interspecies QS. Herein, a search for novel molecular species that will competitively bind with SRH in the LuxS binding site is performed in silico. Computational screening results are then validated in vitro using an optimized LuxS inhibition …

Cytochrome B5 Reductase Encoded By Cbr1 Is Essential For A Functional Male Gametophyte In Arabidopsis, Laura L. Wayne, James G. Wallis, Rajesh Kumar, Jennifer E. Markham, John Browse

Department of Biochemistry: Faculty Publications

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 …