Other Biochemistry, Biophysics, and Structural Biology Commons^™

Toxoplasma Dj-1 Regulates Organelle Secretion By A Direct Interaction With Calcium-Dependent Protein Kinase 1, Matthew A. Child, Megan Garland, Ian Foe, Peter Madzelan, Moritz Treeck, Wouter A. Van Der Linden, Kristina Oresic Bender, Evanthie Weerapana, Mark A. Wilson, John C. Boothroyd, Michael L. Reese

Department of Biochemistry: Faculty Publications

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-A resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, …

Structural And Mechanistic Insights Into Hemoglobincatalyzed Hydrogen Sulfide Oxidation And The Fate Of Polysulfide Products, Victor Vitvitsky, Pramod K. Yadav, Sojin An, Javier Seravalli, Uhn-Soo Cho, Ruma V. Banerjee

Department of Biochemistry: Faculty Publications

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H₂S but, lacking organelles, cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H₂S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human …

Short Carboxylic Acid-Carboxylate Hydrogen Bonds Can Have Fully Localized Protons, Jiusheng Lin, Edwin Pozharski, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homolog YajL was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp23 that satisfies standard donor-acceptor distance criteria for …

Oxidative Stress, Metabolomics Profiling, And Mechanism Of Local Anesthetic Induced Cell Death In Yeast, Cory Honsinger Thomas Boone, Ryan A. Grove, Dana Adamcova, Javier Seravalli, Jiri Adamec

Department of Biochemistry: Faculty Publications

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by …

Secs (Sinusoidal Endothelial Cells), Liver Microenvironment, And Fibrosis, Vaishaali Natarajan, Edward N. Harris, Srivatsan Kidambi

Department of Biochemistry: Faculty Publications

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs …

Gene Duplication And Neo-Functionalization In The Evolutionary And Functional Divergence Of The Metazoan Copper Transporters Ctr1 And Ctr2, Brandon L. Logeman, L. Kent Wood, Jaekwoon Lee, Dennis J. Thiele

Department of Biochemistry: Faculty Publications

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu_ across both the plasma membrane and endomembranes. Although mammalian Ctr¹ functions as a Cu_ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr², has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr¹ activity, but …

The Intestinal Copper Exporter Cua-1 Is Required For Systemic Copper Homeostasis In Caenorhabditis Elegans, Haarin Chun, Anuj Kumar Sharma, Jaekwon Lee, Jefferson Chan, Shang Jia, Byung-Eun Kim

Department of Biochemistry: Faculty Publications

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by …

Structural Determinants Allowing Transferase Activity In Sensitive To Freezing 2, Classified As A Family I Glycosyl Hydrolase, Rebecca Roston, Kun Wang, Leslie A. Kuhn, Christoph Benning

Department of Biochemistry: Faculty Publications

Background: SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance.

Results: Although the active site of SFR2 is identical to hydrolases, adjacent loop regions contribute to its transferase activity.

Conclusion: Transferase activity evolved by modifications external to the core catalytic site.

Significance: Defined structure-function relationships will inform engineering of transferases and freeze tolerance.

Sending Out An Sos: Mitochondria As A Signaling Hub, Iryna Bohovych, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Normal cellular physiology is critically dependent on numerous mitochondrial activities including energy conversion, cofactor and precursor metabolite synthesis, and regulation of ion and redox homeostasis. Advances in mitochondrial research during the last two decades provide solid evidence that these organelles are deeply integrated with the rest of the cell and multiple mechanisms are in place to monitor and communicate functional states of mitochondria. In many cases, however, the exact molecular nature of various mitochondria-to-cell communication pathways is only beginning to emerge. Here, we review various signals emitted by distressed or dysfunctional mitochondria and the stress-responsive pathways activated in response to …

Loss Of Exogenous Androgen Dependence By Prostate Tumor Cells Is Associated With Elevated Glucuronidation Potential, Brenna M. Zimmer, Michelle E. Howell, Qin Wei, Linlin Ma, Trevor Romsdahl, Eileen G. Loughman, Jennifer E. Markham, Javier Seravalli, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD⁺-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate, and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen dependent versus castration resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration resistant tumor cells expressed higher …

Small-Angle X-Ray Scattering Studies Of The Oligomeric State And Quaternary Structure Of The Trifunctional Proline Utilization A (Puta) Flavoprotein From Escherichia Coli, Ranjan K. Singh, John D. Larson, Weidong Zhu, Robert P. Rambo, Greg L. Hura, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon.

Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization.

Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs.

Significance: The first three-dimensional structural information for any trifunctional PutA is reported.

Methods For Detecting Microbial Methane Production And Consumption By Gas Chromatography, Jared T. Aldridge, Jennie L. Catlett, Megan L. Smith, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic …

Stabilin-1 And Stabilin-2 Are Specific Receptors For The Cellular Internalization Of Phosphorothioate-Modified Antisense Oligonucleotides (Asos) In The Liver, Colton M. Miller, Aaron J. Donnerr, Emma E. Blank, Andrew W. Egger, Brianna M. Kellar, Michael E. Østergaard, Punit P. Seth, Edward N. Harris

Department of Biochemistry: Faculty Publications

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro^TM, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform …

Nitrogenase Femoco Investigated By Spatially Resolved Anomalous Dispersion Refinement, Thomas Spatzal, Julia Schlesier, Eva-Maria Burger, Daniel Sippel, Limei Zhang, Susana L.A. Andrade, Douglas C. Rees, Oliver Einsle

Department of Biochemistry: Faculty Publications

The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S 1⁄4 3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the …

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 …