bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering Potentiated Hsp104 Variants With Enhanced Substrate‐Specificity to Counter Neurodegeneration
Authors: Mack Korrie L. , Shorter James 
Published: November 2023

Hsp104 is a hexameric AAA+ ATPase and disaggregase found in yeast. Together with the Hsp70 chaperone system, Hsp104 uses energy from ATP hydrolysis to reactivate and refold protein substrates. Hsp104 eliminates aggregates that accumulate after environmental stress, and with the exception of the Hsp110 system, is the only protein known to rapidly disassemble amyloid. Interestingly, metazoa lack an Hsp104 homolog, and as such, humans have limited ability to eradicate amorphous aggregates and amyloid. This shortcoming is problematic, as protein misfolding underlies several neurodegenerative diseases. As such, Hsp104 is an appealing exogenous candidate for therapeutic targeting of toxic misfolded proteins. We previously engineered potentiated Hsp104 variants that robustly disaggregate and suppress the toxicity of neurodegenerative disease substrates α‐synuclein (implicated in Parkinson's disease) and TDP‐43 and FUS (implicated in amyotrophic lateral sclerosis). The potentiated variants also restore proper cellular localization of these neurodegenerative disease substrates. Yet, potentiated Hsp104 variants lack strong substrate specificity, an important attribute needed to prevent nonspecific unfolding of essential proteins. Here, we developed substrate‐specific variants by rationally tuning conserved Hsp104 pore loop residues, Y257 and Y662, which are known to contact substrate. In altering the pore loops in potentiated backgrounds, we discovered variants specific for α‐synuclein. In yeast proteinopathy models, the α‐synuclein‐specific variants suppress toxicity of α‐synuclein, and only minimally suppress toxicity of TDP‐43 and FUS. The α‐synuclein‐specific variants are inherently less toxic to yeast than generally potentiated variants, and prevent α‐synuclein‐induced neurodegeneration in a C. elegans model of Parkinson's disease. Interestingly, we observed differences between the α‐synuclein‐specific variants in their ability to suppress toxicity and restore proper cellular localization of α‐synuclein. We have also uncovered a new set of generally potentiated Hsp104 variants that strongly suppress toxicity of α‐synuclein, TDP‐43, and FUS. Overall, our findings expose crucial insights to tailoring Hsp104 to selectively and therapeutically target toxic misfolded species.Support or Funding InformationNSF Graduate Research Fellowship (DGE‐1321851)
Redox modification of Fes1 and its role in cellular oxidative stress response
Authors: Nicklow Erin , Sevier Carolyn 
Published: November 2023

Elevated levels of reactive oxygen species (ROS) are associated with many degenerative disorders (like aging, Alzheimer's, atherosclerosis, and diabetes). Increased cellular ROS levels correlate with accumulation of irreversibly oxidized proteins, which are prone to misfolding and aggregation. 70‐kDa heat shock proteins (Hsp70s) are a conserved family of ATPases that serve as molecular chaperones to refold misfolded and aggregated proteins, such as those damaged by ROS. As such, Hsp70s play a critical role in protein quality control under oxidative stress.To examine the role of cytosolic Hsp70s in coping with oxidative stress, I screened S. cerevisiae strains mutated for each of the chaperones and their cofactors for peroxide sensitivity. Notably, I found that the nucleotide exchange factor Fes1 is required for cell survival during oxidative stress, and that when cells are grown in medium containing peroxide, Fes1 is modified directly by ROS. Preliminary data suggest that this oxidative modification of Fes1 alters its activity, which I hypothesize confers a protective role during stress conditions and allows cells to sense and respond to elevated ROS levels. I am currently focused on characterizing this event to determine how Fes1 is modified by ROS and how modification affects Fes1's protein quality control activities in the cell.
Protein‐Based Models of Nickel Metalloenzymes
Authors: Shafaat Hannah S. , Manesis Anastasia C. , Schneider Camille R. , O'Connor Matthew C. 
Published: November 2023

Nickel‐containing metalloenzymes are highly relevant in the context of biological energy conversion reactions. Of particular interest to our group is the carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) enzyme, which is responsible for the synthesis of acetyl‐CoA from CO2. This metabolic pathway is one of the oldest mechanisms by which cells convert inorganic carbon into biomass and has been implicated in the chemoautotrophic origin of life theory. However, due to the significant complexity and fragility of the native CODH/ACS complex, the mechanistic details underlying these reactions remain poorly understood.We seek to address this disparity using existing metalloprotein platforms to develop active‐site models for C1 conversion reactions. This approach allows targeted engineering of an isolated active site while retaining the complexity of an outer coordination sphere, recognized as critical for achieving the high levels of activity seen in native enzymes. Using azurin as a prototype model protein scaffold, we have been able to generate structural and functional mimics of CODH and ACS. Comprehensive characterization of these systems using spectroscopy, electrochemistry, structural investigations, computational results, and activity assays reveals key similarities and mechanistic distinctions between the model proteins and the native enzyme. Ongoing efforts to optimize activity using targeted mutations in the primary and secondary coordination spheres will be discussed. Ultimately, these minimal enzyme models demonstrate potential to achieve reactivity approaching that of native CODH/ACS.
Salt‐Dependent Protein Splicing of the Intein in the Haloquadratum walsbyi DNA Polymerase
Authors: Reidy Deirdre Ann 
Published: November 2023

Protein splicing is a post‐translational process by which an intervening polypeptide, or intein, can excise itself and ligate the flanking polypeptides, or exteins. Two extreme halophilic archaea, Halobacterium salinarum (Hsa) and Haloquadratum walsbyi (Hwa), have proteins with inteins. Previous work in the lab demonstrated that splicing of the Hsa DNA Polymerase II (Polll) intein is salt‐dependent. We attempted to compare the homologous PolII‐a intein in Hwa. However, the protein was insoluble when expressed in two different strains of E. coli and with addition of salt. Currently, we are attempting to increase the solubility of Hwa PolII‐a in order to compare its splicing activity to that of Hsa PolII. We are also interested in the activity of a second Hwa PolII intein, PolII‐b. Additionally, we are interested in studying the influence of salt on the homing endonuclease activities of the Hwa PolII‐a and PolII‐b inteins.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM), the Dreyfus Foundation (KVM), and William F. McCall Jr. (DAR).
Inhibition of Acetyl Cholinesterase Extracted from Five Parts of the Brain by 5,6 Dihydroxytryptamine
Authors: Osman Mohamed Yousry Hassan 
Published: November 2023

5,6 dihydroxytryptamine (DHT) is a neurogenic analogue of serotonin and physostigmine (eserine), both containing tertiary nitrogen group which is lipid soluble. The addition of 5,6 DHT to cholinesterase (ChE) extracted from whole and five different parts of rat brain namely: basal ganglia, cerebellum, frontal cortex, medulla oblongata, and pons in vitro was studied. The inhibition of ChE in the different parts of the brain was of the competitive type. The highest values of the enzyme‐inhibitor dissociation constant (Ki) were obtained with the extracts of medulla oblongata and pons. Those are the parts responsible for the vital centres. The inhibition of the extracts by DHT was reversible and the enzymes restored their whole activities by dialysis. The difference in the inhibition between the extracts of the parts was due to the difference in the gray and white matter content in these parts which was proved experimentally in this work. The inhibition was higher in the extracts obtained from the parts containing white matter than those containing gray matter. Erythrocyte and plasma ChEs were also inhibited by DHT. The inhibition of erythrocyte ChE was higher than plasma ChE and lower than the inhibition of the extracts of the brain parts. The inhibition of ChE by 5,6 DHT saves acetylcholine which helps in certain diseases like myasthenia gravis, Alzheimer's disease and Parkinsonism.Support or Funding InformationDepartment of Biochemistry, Medical Research Institute, Alexandria University,165 Horria Avenue Elhadarah, Alexandria, EGYPT
Defining energetic homeostasis in Toxoplasma gondii
Authors: Murphy Robert D , Dhara Animesh , Sinai Anthony P , Gentry Matthew S 
Published: November 2023

Toxoplasma gondii is an opportunistic, protozoan parasite that infects roughly one‐third of humans worldwide. Shortly after infection, T. gondii establishes cysts, occupied by the slow‐growing bradyzoite stage in the CNS or muscle tissue where it will persist for the lifetime of the host. Bradyzoite biology is poorly understood, especially in regards to energetics. Accumulation of insoluble carbohydrate structures known as amylopectin granules (AGs) in the cytoplasm is a distinguishing morphological feature of bradyzoites, and thus suggests a potential source of energetic and biosynthetic potential. We have identified multiple enzymes involved in maintaining energetic homeostasis in the T. gondii. We have characterized these enzymes using bioinformatics and biochemical approaches. These studies seek to characterize this set of enzymes as gatekeepers to accessing the metabolic potential stored in AGs.Support or Funding InformationThis project is funded by the IDeA award from NIH/NIGMS 5P30GM110787 (COBRE for the Center for Molecular Medicine. PI Louis B Hersh, University of Kentucky) through the University of Kentucky Center for Molecular Medicine.
Measuring the Positioning and Translocation of the Swinging‐Arm Domain of Pyruvate Carboxylase
Authors: Hakala Joshua H , St. Maurice Martin 
Published: November 2023

A central goal of studies on large, multi‐domain enzymes such as polyketide synthases and fatty acid synthase is to reveal the mechanism(s) contributing to carrier domain translocation and coordination. Pyruvate carboxylase (PC) is a multi‐domain enzyme with a swinging‐arm domain that coordinates reactions between two remote active sites and can serve as a structurally and biophysically accessible paradigm to study multicomponent swinging‐domain enzymes. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. This serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. The biotin cofactor on the biotin carrier domain (BCCP) is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then swings to the carboxyltransferase (CT) domain where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate. These reactions, which take place in distantly located active sites, are tightly coordinated under optimal reaction conditions. In order to better understand the mechanism of coordination, the equilibrium positioning and translocation of the swinging BCCP domain was directly observed using site‐specific cross‐linking and fluorescence anisotropy. Probes were positioned at the CT active site and the so‐called “exo‐binding site”, permitting a detailed analysis of BCCP domain positioning and movement in response to various substrates and allosteric effectors. These studies have revealed that substrates and effectors shift the BCCP domain binding equilibrium from one domain to another and offer the first evidence that the carrier domain exists in a dynamic equilibrium between active sites.Support or Funding InformationThis work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01.
Deciphering the Logic of Natural Product Biosynthesis
Authors: Li Bo 
Published: November 2023

Natural products and derivatives constitute a large fraction of modern medicine, especially in the arena of antimicrobial therapy. To identify and engineer new natural products, it is critical to understand the mechanisms of chemical transformations responsible for biosynthesis. My group seeks to characterize the novel enzyme chemistries in the construction of biosynthetic building blocks and those that assemble these building blocks into complex and potent natural products. In this talk, I will discuss our work on the biosynthesis of thiomarinol that is a naturally occurring hybrid antibiotic between two antimicrobial “warheads”, pseudomonic acid and holothin, linked by an amide bond. We investigated the enzyme basis of “hybridity” in thiomarinol biosynthesis (1). Contrary to the previous proposal, we showed that a CoA ligase and an acyltransferase act in tandem to catalyze the amidation step. We also studied the substrate scope of this enzyme pair in vitro and demonstrated that these enzymes can incorporate non‐native functional groups into thiomarinol analogs. Our results revealed important insight regarding convergent evolution and uncovered a useful strategy to create new “hybrid” molecules. I will also discuss our ongoing efforts on the biosynthesis of nonproteinogenic amino acids, important building blocks for natural product biosynthesis. Mechanistic insights gained from our research will enable identification and engineering of natural products as potential drug leads.Support or Funding InformationNational Institute of Health GM099904‐04
Inhibition of Tumor Necrosis Factor Receptor 1 Signaling by Small Molecules
Authors: Lo Chih Hung , Vunnam Nagamani , Lewis Andrew , Chiu Ting‐Lan , Brummel Benjamin , Schaaf Tory , Grant Benjamin , Bawaskar Prachi , Thomas David , Sachs Jonathan 
Published: November 2023

Tumor necrosis factor receptor 1 (TNFR1) is a member of the TNFR superfamily which can be activated by binding to one of two cognate ligands, tumor necrosis factor (TNF‐α) or lymphotoxin‐α (LT‐α), via its extracellular domain. Ligand binding leads to network formation and signaling cascade of IκBα degradation and NF‐κB activation, resulting in inflammatory and autoimmune diseases. Current treatments, such as etanercept and infliximab, reduce symptoms through sequestration of free ligands. However, these treatments are expensive and induce dangerous side‐effects due to off‐target inhibition of other related TNF receptors that are not involved in the disease condition. As a consequence, there is a desperate need for receptor‐specific treatments, which hold the promise of overcoming the limitations of conventional therapeutics. It has been proposed that TNFR1 pre‐ligand assembly domain (PLAD), a portion of the extracellular region of TNFR1 that mediates receptor‐chain association essential for signaling, is an important therapeutic target in inflammatory arthritis. This motivates the discovery of small‐molecule inhibitors that could bind to TNFR1 PLAD to disrupt receptor‐receptor interactions and inhibit downstream signaling. In this study, we have engineered a biosensor exhibiting fluorescence resonance energy transfer (FRET) by attaching fluorophores to the intracellular domain of TNFR1. By using a fluorescence lifetime screening platform, two hit compounds were identified to inhibit TNFR1 function. These small‐molecule inhibitors reduce FRET and disrupt PLAD‐PLAD interaction without ablating ligand binding. The biosensor and the fluorescence lifetime screening approach reported here provide a very promising high‐throughput drug discovery platform for TNF receptors, as well as oligomeric receptors in general.Support or Funding InformationThis work is supported by National Institutes of Health Grant R01 GM107175 and R42 DA037622
Assessing the Effects of ErbB2 Kinase Inhibition in Conjunction with Broad Spectrum Anti‐Cancer Treatments
Authors: Tremper Catherine , Matha Amber Rae , Taylor Christopher Edward 
Published: November 2023

The ErbB family of tyrosine kinases drives cell growth and has been associated with increased malignancy and resistance to treatment. In particular, erbB2 has been identified as an important target for chemotherapy. Although several inhibitors of erbB kinases are clinically available, there is a continued need to identify treatment combinations that improve the efficacy of these inhibitors and slow the development of resistance.Our research examines the effects of combining erbB2 kinase inhibitors with bortezomib (a protease inhibitor) and panobinostat (an HDAC inhibitor), both of which are broadly effective against cancers, including those which overexpress erbB2. This presentation will compare the effects of combination treatments relative to the effects of each individual agent, using current methods for examining synergy such as the Bliss Independence Model, and Loewe's isobologram based model.
Effect of bleaching products on proteins of teeth
Authors: Frazier Randy , Panah Abdullah , Keenan Kelly 
Published: November 2023

Americans spend an estimated 1.4 billion dollars per year on over‐the‐counter bleaching products which whiten teeth. The active ingredient is hydrogen peroxide which can damage proteins. The goal of this project was to examine the effect of bleaching on the proteins in teeth. Extracted teeth were either not bleached at all or received either one or two rounds of bleaching with a commercial product. Non collagen proteins in teeth were extracted using EDTA and protein content was measured using the Lowry procedure. The extracted proteins were then separated into individual components using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and the molecular weights were compared to those of proteins known to be in the enamel or dentin of teeth. To further characterize these proteins, they were extracted from the gels, hydrolyzed and the amino acids were identified using reverse phase high performance liquid chromatography (RP HPLC). Results do show a reduction in the amount of protein following bleaching.Support or Funding InformationThe authors wish to thank the NAMS and Provost Travel Funds and the Provost Opportunity Funds.
Therapeutic targeting of MLL degradation pathways in MLL‐rearranged leukemia
Authors: Liang Kaiwei , Volk Andrew G. , Haug Jeffrey S. , Marshall Stacy A. , Woodfin Ashley R. , Bartom Elizabeth T. , Gilmore Joshua M. , Florens Laurence , Washburn Michael P. , Sullivan Kelly D. , Espinosa Joaquin M. , Cannova Joseph , Zhang Jiwang , Smith Edwin R. , Crispino John D. , Shilatifard Ali 
Published: November 2023

Chromosomal translocations of the mixed‐lineage leukemia (MLL) gene with various partner genes result in aggressive MLL leukemia with dismal outcomes. Despite relatively equal contributions of the rearranged allele and the remaining wild‐type allele at the mRNA level, the MLL chimeric protein is much more stable than the wild‐type MLL protein. Here, we used Multidimensional Protein Identification Technology (MudPIT) and established a pooled genome‐wide shRNA library screen to identify the factors and pathways involved in regulating the stability of the wild‐type MLL protein. Targeting wild‐type MLL degradation, through blocking of these pathways and factors, preferentially impedes MLL leukemia cell proliferation and downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the Super Elongation Complex (SEC), as revealed by RNA sequencing and Chromatin Immunoprecipitation Sequencing (ChIP‐Seq). Stabilizing wild‐type MLL protein by inhibition of these pathways displaces the MLL chimera from some of its target genes and therefore relieves the cellular oncogenic addiction to MLL chimeras. Furthermore, pharmacologically inhibition of these pathways with small molecules substantially delays progression and improves survival of murine MLL‐AF9 leukemia in vivo. Therefore, disrupting the balance between wild‐type MLL and MLL chimeras by stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers driven by chromosomal translocations.Support or Funding InformationThis study was supported by the Samuel Waxman Cancer Research Foundation to John D. Crispino and National Institute of Health grants, CA211428 to Edwin R. Smith, CA117907 to Joaquin M. Espinosa, CA101774 to John D. Crispino. and R35CA197569 to Ali Shilatifard
Optimization of Reconstituted High Density Lipoprotein (rHDL) nanoparticles (NPs) for Short‐Interfering RNA (siRNA) Delivery
Authors: Mooberry Linda , Sabnis Nirupama , Lacko Andras 
Published: November 2023

ObjectiveThe objective of these studies was to characterize siRNA transporting rHDL NPs and to evaluate their suitability for translation toward clinical applications. Previous studies have shown that rHDL NPs are capable of promoting the delivery and retention of short‐interfering RNA (siRNA) by cancer cells and tumors (Shahzad et al., 2011).MethodsCholate dialysis and a facilitated mixing process through a microfluidics instrument (Precision Nanosystems NanoAssemblr) were used to prepare the siRNA containing rHDL NPs. Diameters of the NPs were estimated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Stability of the rHDL/siRNA formulations were examined before and after lyophilization. Western blotting was used to assess the expression of the transcription factor STAT‐3 in the SKOV‐3 ovarian cancer cell line following siRNA treatment.ResultsA series of neutralizers were evaluated to suppress the negative charges of siRNA. Oligolysine (MW=500–2000) was found to be most effective with an incorporation efficiency of 88.8 ± 10.1% by cholate dialysis and 88.6 ± 2.0% by microfluidics. The size of the complexes estimated by DLS was found to be 59.6 ± 6.7 nm for nanoparticles prepared by cholate dialysis and 39.6 ± 6.3 nm for NPs prepared by microfluidics. However, TEM images revealed uniform, spherical NPs with a diameter of 23.9 nm ± 2.9. Examination of diameter after lyophilization with a cryoprotective agent showed only a 6.8 ± 2.5 % increase in the size of the nanoparticle formulation with 90.5 ± 3.2% retention of the nucleic acid. The effective knockdown of the expression of the STAT‐3 target was achieved in an ovarian cancer (SKOV‐3) cell line.ConclusionsThis study is the first to report the preparation of NPs with a protein component, using the NanoAssemblr instrument. This rHDL/siRNA formulation has a small diameter, good efficiency at incorporating siRNA, and is biologically active. The siRNA containing rHDL NPs were found to be stable after lyophilization, with little change in diameter and siRNA content.Support or Funding InformationThe authors' research is being supported by Cancer Prevention and Research Institute of Texas, Wheels for Wellness, Fort Worth TX and the Rutledge Foundation.