Biochemistry Commons^™

Definition Of The Landscape Of Chromatin Structure At The Frataxin Gene In Friedreich’S Ataxia, Eunah Kim

Dissertations and Theses (Open Access)

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA …

Planning Combinatorial Disulfide Cross-Links For Protein Fold Determination, Fei Xiong, Alan M Friedman, Chris Bailey-Kellogg

Dartmouth Scholarship

Fold recognition techniques take advantage of the limited number of overall structural organizations, and have become increasingly effective at identifying the fold of a given target sequence. However, in the absence of sufficient sequence identity, it remains difficult for fold recognition methods to always select the correct model. While a native-like model is often among a pool of highly ranked models, it is not necessarily the highest-ranked one, and the model rankings depend sensitively on the scoring function used. Structure elucidation methods can then be employed to decide among the models based on relatively rapid biochemical/biophysical experiments.

Biochemical And Pharmacological Characterization Of Cytochrome B5 Reductase As A Potential Novel Therapeutic Target In Candida Albicans, Mary Jolene Patricia Holloway

USF Tampa Graduate Theses and Dissertations

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics.

We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as …

Investigating A Conformational Change In The Enzyme Neurolysin, Fei Xiong

Kaleidoscope

No abstract provided.

Investigating Calmodulin-Long Qt Syndrome Restorative Interactions Through Combinatorial Approaches, Michael Bricken

Kaleidoscope

No abstract provided.

Boron Stress Activates The General Amino Acid Control Mechanism And Inhibits Protein Synthesis, Irem Uluisik, Alaattin Kaya, Dmitri E. Fomenko, Huseyin C. Karakaya, Bradley A. Carlson, Vadim N. Gladyshev, Ahmet Koc

Department of Biochemistry: Faculty Publications

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a …

A Lipid-Anchored Snare Supports Membrane Fusion, Hao Xu, Michael Zick, William T. Wickner, Youngsoo Jun

Dartmouth Scholarship

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes …

The Crosstalk Between Human Fatty Acid Transport Protein 1 And Fatty Acid Transport Protein 4, Zhe Yuan

Department of Biochemistry: Dissertations, Theses, and Student Research

Fatty acid transport proteins (FATPs) provide pivotal roles in fatty acid transport and activation and thus are crucial for overall fatty acid homeostasis. Peroxisome Proliferators Activated Receptors (PPARs) are important transcription factors, which control many genes that govern lipid metabolism.

Using 293 T-REx cell lines that stably express FATP1 or FATP4 from a tetracycline-inducible promoter, this work evaluated gene expression of key genes involved in fatty acid metabolism using QPCR, protein expression of FATP1 and FATP4 using Western blots, and fatty acid transport to address the roles of these two FATP isoforms in fatty acid homeostasis. This work found that …

Total Synthesis Of 4'-Ester Resveratrol Analogs And 8.9-Amido Geldanamycin Analog And Toward The Total Synthesis Of (-)-Englerin A, Yong Wang

Theses and Dissertations

Total Synthesis of 4'-ester Resveratrol Analogs and 8, 9-amido Geldanamycin Analog and toward the Total Synthesis of (-)-Englerin A Yong Wang Department of Chemistry and Biochemistry, BYU Doctor of Philosophy The phytoalexin resveratrol and its 4'-ester analogs have been prepared with a decarbonylative Heck reaction. The deprotecting step has been modified and improved to increase yield and avoid chromatography. A set of resveratrol analogs and resveratrol have been tested with melanoma and pancreatic cell assays. The 8, 9-amido Geldanamycin analog has been synthesized with a convergent route, involving 28 simplified steps in its longest linear sequence. Synthetic methodologies, such as …

Using Vesicular Stomatitis Virus Glycoprotein To Pseudotype Bacmam Viruses For Enhancement Of Transduction Efficiency, Lydia Sanchez

PCOM Biomedical Studies Student Scholarship

Baculovirus has been a common tool for protein production since the mid 1980s. The virus has an exclusive tropism only capable of infecting the larval stages of insects. Originally, baculovirus recombinant gene expression used Spodoptera frugiperda (Sf9) insect cells as the host cells for protein production. However, in 1995, it was discovered that baculoviruses could be modified to transduce mammalian cells with the insertion of a mammalian promoter and gene of interest. Due to its ease of generation and low biology safety hazards, this technology, known as BacMam (Baculovirus Mammalian) is popular in the research field for protein production and …

Iron Insufficiency Compromises Motor Neurons And Their Mitochondrial Function In Irp2-Null Mice, Suh Young Jeong, Daniel R. Crooks, Hayden Wilson-Ollivierre, Manik C. Ghosh, Rachid Sougrat, Jaekwon Lee, Sharon Cooperman, James B. Mitchell, Carole Beaumont, Tracey A. Rouault

Department of Biochemistry: Faculty Publications

Genetic ablation of Iron Regulatory Protein 2 (Irp2, Ireb2), which post-transcriptionally regulates iron metabolism genes, causes a gait disorder in mice that progresses to hind-limb paralysis. Here we have demonstrated that misregulation of iron metabolism from loss of Irp2 causes lower motor neuronal degeneration with significant spinal cord axonopathy. Mitochondria in the lumbar spinal cord showed significantly decreased Complex I and II activities, and abnormal morphology. Lower motor neurons appeared to be the most adversely affected neurons, and we show that functional iron starvation due to misregulation of iron import and storage proteins, including transferrin receptor 1 …

Akap2 Anchors Pka With Aquaporin-0 To Support Ocular Lens Transparency, Matthew G. Gold, Steve Reichow, Susan E. O'Neill, Chad R. Weisbrod, Lorene K. Langeberg, James E. Bruce, Tamir Gonen, John D. Scott

Chemistry Faculty Publications and Presentations

A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a …

Advances In Structural And Functional Analysis Of Membrane Proteins By Electron Crystallography, Goragot Wisedchaisri, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane …

Exploring The Roles Of Nucleobase Desolvation And Shape Complementarity During The Misreplication Of O6-Methylguanine, Delia Chavarria, Andrea Ramos Serrano, Ichiro Hirao, Anthony J. Berdis

Chemistry Faculty Publications

O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a …

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Beta-Lysine Discrimination By Lysyl-Trna Synthetase, Marla S. Gilreath, Hervé Roy, Tammy J. Bullwinkle, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P is modified with (R)‐β‐lysine by the lysyl‐tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α‐ and β‐lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α‐lysine, while the G469A and A233S/G469A variants decreased stable α‐lysyl‐adenylate formation. A233S LysRS recognized β‐lysine better than wildtype, suggesting a role for this residue in discriminating α‐ and β‐amino acids. Both enantiomers of β‐lysine were substrates for tRNA aminoacylation by LysRS, which, together with …

Follicular Dendritic Cells, Resting Cd4+ T Cells And Human Immunodeficiency Virus Expression, Changna Wang

Theses and Dissertations

Many events associated with Human Immunodeficiency Virus (HIV) infection/replication occur in and around the germinal centers (GCs) of secondary lymphoid tissues where follicular dendritic cells (FDCs) reside, suggesting that this microenvironment may contribute unique signaling that is important to viral progression. My research focused on characterizing signaling, both positive and negative, contributed by FDCs that affects HIV infection and replication. Specifically, I determined if FDC signals could induce the expression of latent HIV in T cells and if so, to characterize the signaling pathways involved. Moreover, I also examined the ability of FDCs to produce inhibitory signals that might block …

Stomatin-Like Protein 2 Binds Cardiolipin And Regulates Mitochondrial Biogenesis And Function., Darah Christie, Caitlin D Lemke, Isaac M Elias, Luan A Chau, Mark G Kirchhof, Bo Li, Eric H Ball, Stanley D Dunn, Grant M Hatch, Joaquín Madrenas

Biochemistry Publications

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched …

Single Molecule Analysis Of The Arabidopsis Fra1 Kinesin Shows That It Is A Functional Motor Protein With Unusually High Processivity, Chuanmei Zhu, Ram Dixit

Biology Faculty Research

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)–GFP). We found that FRA1(707)–GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated …

The Worlds Of Splicing And Chromatin Collide, J. Adam Hall, Philippe T. Georgel

Biological Sciences Faculty Research

Both transcription and splicing take place in a nuclear environment which, at face value, may seem refractory to the efficiency afforded by the coupling of both processes. This environment, chromatin, was once viewed as only a passive packaging system for genetic material, with very little contribution to the variety of nuclear activities occurring within and around it. However, overwhelming evidence now points to the chromatin environment as being highly dynamic, and an active player in nuclear activities.