Other Biochemistry, Biophysics, and Structural Biology Commons^™

Ef-P Dependent Pauses Integrate Proximal And Distal Signals During Translation, Sara Elgamal, Assaf Katz, Steven J. Hersch, David Newsom, Peter White, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at …

Trnas As Regulators Of Biological Processes, Medha Raina, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Transfer RNAs (tRNA) are best known for their role as adaptors during translation of the genetic code. Beyond their canonical role during protein biosynthesis, tRNAs also perform additional functions in both prokaryotes and eukaryotes for example in regulating gene expression. Aminoacylated tRNAs have also been implicated as substrates for non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosyntheses. Most recently tRNA fragments, or tRFs, have also been recognized to play regulatory roles. Here, we examine in more detail some of the new functions emerging for tRNA in a variety of cellular processes …

Oxidation Of Cellular Amino Acid Pools Leads To Cytotoxic Mistranslation Of The Genetic Code, Tammy J. Bullwinkle, Noah M. Reynolds, Medha Raina, Adil Moghal, Eleftheria Matsa, Andrei Rajkovic, Huseyin Kayadibi, Farbod Fazlollahi, Christopher Ryan, Nathaniel Howitz, Kym F. Faull, Beth A. Lazazzera, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress …

Implications Of The Landauer Limit For Quantum Logic, F. Matthew Mihelic

Faculty Publications

The design of any system of quantum logic must take into account the implications of the Landauer limit for logical bits. Useful computation implies a deterministic outcome, and so any system of quantum computation must produce a final deterministic outcome, which in a quantum computer requires a quantum decision that produces a deterministic qubit. All information is physical, and any bit of information can be considered to exist in a physicality represented as a decision between the two wells of a double well potential in which the energy barrier between the two wells must be greater than kT·ln2. Any proposed …

Reduced Amino Acid Specificity Of Mammalian Tyrosyl-Trna Synthetase Is Associated With Elevated Mistranslation Of Tyr Codons, Medha Raina, Adil Moghal, Amanda Kano, Mathew Jerums, Paul D. Schnier, Shun Luo, Rohini Deshpande, Pavel D. Bondarenko, Henry Lin, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, …

A Course-Based Research Experience: How Benefits Change With Increased Investment In Instructional Time, Christopher D. Shaffer, Cheryl Bailey, Students And Tas In Genomics Education Partnership Courses

Department of Biochemistry: Faculty Publications

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge.We have developed a bioinformatics project that provides a coursebased research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment …

Structures Of The Puta Peripheral Membrane Flavoenzyme Reveal A Dynamic Substrate-Channeling Tunnel And The Quinone-Binding Site, Harkewal Singh, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004- residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal …

Genome-Wide Rnai Ionomics Screen Reveals New Genes And Regulation Of Human Trace Element Metabolism, Mikalai I. Malinouski, Nesrin M. Hasan, Yan Zhang, Javier Seravalli, Jie Lin, Andrei Avanesov, Svetlana Lutsenko, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Trace elements are essential for human metabolism and dysregulation of their homoeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires …

The Abcs Of The Ribosome, Kurt Fredrick, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

An ABC protein that binds the ribosomal exit site suggests a new mechanism for direct regulation of translation in response to changing ATP levels in the cell.

Arabidopsis Accelerated Cell Death 11, Acd11, Is A Ceramide-1-Phosphate Transfer Protein And Intermediary Regulator Of Phytoceramide Levels, Dhirendra K. Simanshu, Xiuhong Zhai, David Munch, Daniel Hofius, Jennifer E. Markham, Jacek Bielawski, Alicja Bielawska, Lucy Malinina, Julian G. Molotkovsky, John W. Mundy, Dinshaw J. Patel, Rhoderick E. Brown

Department of Biochemistry: Faculty Publications

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1- phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center …

Biochemical Analysis Of Blood Of Native Cattle In The Hilly Area Of Bangladesh, M. A. Mamun, M. M. Hassan, A. H. Shaikat, S.K.M.A. Islam, M. A. Hoque, M. Uddin, M. B. Hossain

Biochemistry Collection

A yearlong study was carried out in indigenous cattle of five upazila namely Khagrachari Sadar, Laxmichari, Matiranga, Mahalchari and Dighinala in Khagrachari Hill District, Bangladesh for determination of statuses of various biochemical parameters. Apparently healthy cattle (100) were selected and blood samples were collected aseptically. Biochemical analysis of carbohydrates, proteins, fats, minerals, electrolytes and enzymes were performed by using automated biochemical analyzer (Humalizer®-3000). The results showed that, the variations of parameters were not significant (P>0.05) between male and female. Although there was a slight increase of calcium 8.01±0.32mg/dl, glucose 69.21±2.31mg/dl, LDL 5.69±0.22mg/dl and uric acid 3.85±0.34mg/dl of female compared …

Book Review Of Dane Kennedy, The Last Blank Spaces: Exploring Africa And Australia, Adrian S. Wisnicki

Department of Biochemistry: Faculty Publications

Mid-nineteenth-century British exploration, particularly the stories of the “heroic” individuals who carried out this exploration, remains a topic of worldwide interest, as most recently evidenced by the many events in Britain and Africa celebrating the 2013 bicentenary of David Livingstone’s birth. Dane Kennedy’s intriguing study takes issue with such readings of the historical record by foregrounding an epistemological tension that lies at the heart of nineteenthcentury British exploration discourse and practice. The book examines the collision—as it played out in the exploration of African and Australia—between metropolitan scientific protocols and non-Western quotidian realities. Nineteenth-century explorers, argues Kennedy, left home with …

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 …

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 …

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 …

Evidence For Hysteretic Substrate Channeling In The Proline Dehydrogenaseand ∆1-Pyrroline-5-Carboxylate Dehydrogenase Coupled Reaction Of Proline Utilizationa(Puta), Michael A Moxley, Nikhilesh Sanyal, Navasona Krishnan, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism.

Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step.

Conclusion: Substrate channeling in PutA exhibits hysteretic behavior.

Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation.

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 …

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 …

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 …

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 …