bluuebunny/crossref_metadata_2025_split huggingface dataset:
Probing the role of allostery in Rho and Ras GTPases
Authors: Marcus Kendra , Ma Buyong , Nussinov Ruth , Mattos Carla 
Published: November 2023

The Ras superfamily of GTPases is a family of binary molecular switches that regulate a variety of cellular processes, such as proliferation and morphological control. Many members of the Ras superfamily are localized to membrane environments and interact with a variety of effectors to promote unique signaling consequences. Due to the strong correlation of Ras GTPases to cancer, the structural, biophysical, and functional relationships of Ras subfamily GTPases have been extensively studied. In particular, our work has identified functionally‐related networks of allosteric communication across the G domain of HRas. Several networks involve water‐mediated connectivity from the active site of the HRas to remote surfaces of the protein that are proposed to interact with the plasma membrane. Like Ras GTPase subfamily members, Rho GTPases are localized within a plasma membrane environment, but contain an inserted helical region located near its active site. We are currently investigating whether these networks are also important in the Rho subfamily of GTPases, with focus on family member RhoA. We have obtained crystal structures of WT RhoA and of two of its mutants, R70A and E102A. These mutants are located within structurally analogous regions of HRas allosteric communication. The solved structures suggest that these mutants promote crystal contacts along switch II of RhoA, an important surface for effector binding. Distal regions of the protein, including the insert helix, also appear to be perturbed. We have also performed accelerated molecular dynamics (aMD) simulations starting with the X‐ray structures for wild type and mutant RhoA. The extended trajectory of the RhoA models (200 ns) highlight the dynamic role of the insert region towards global communication across the RhoA protein. Validated through residue cross‐correlation analysis, both mutants also hinder communication throughout the GTPase, radiating from the active site towards membrane‐interacting regions. These data suggest that global allosteric communication may be a common feature of the Ras superfamily of small GTPases.Support or Funding InformationThis work is sponsored by a grant from the National Science Foundation (MCB‐1517295)
Purification and Structural Analysis of an Uncharacterized Lytic Protein in Epstein‐Barr Virus
Authors: Scheidt Michael D , Gorres Kelly 
Published: November 2023

Epstein‐Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis. EBV is associated with human cancers, such as Burkitt lymphoma and nasopharyngeal carcinoma, due to infection of the B and epithelial cells respectively. The lytic cycle is known as the productive phase of the viral life cycle; in other terms, when the genome of the virus is undergoing transcription and new virions are being produced within a host cell. In the virion, the viral DNA is enclosed by an ordered protein capsid, which is surrounded by a layer of proteins called the tegument. This project studies an Epstein‐Barr virus tegument protein of unknown function. When the gene encoding the uncharacterized tegument protein is knocked out, virion production is not observed.Though important, the structure and function of this lytic tegument protein has yet to be determined. This project has produced pure samples of this tegument protein for study of the basic structural characteristics. The histidine‐tagged protein was produced in E. coli, including C41(DE3) and SHuffle cells. Both strains of cells contain components to promote foreign protein folding, though SHuffle cells contain chaperones specific to disulfide bond formation. When induced in C41(DE3) competent cells, signs of protein degradation were observed on a reducing SDS‐PAGE gel. Successful induction of full‐length protein was observed in SHuffle cells, indicating potential presence of disulfide bonds in the tertiary structure of the protein. The induced protein was purified by Ni2+‐affinity chromatography. Circular dichroism and dynamic light scattering analysis will be conducted to observe structural characteristics in the protein's native state with the goal of determining the structure and predicting a function for this EBV lytic protein.Along with functioning within the lytic cycle of EBV, this tegument protein has potential ties to multiple sclerosis (MS), a central nervous system disease that causes demyelination and degeneration of neurons. From published studies, it has been found that patients with MS have a much greater antigenic response towards one of the tegument proteins. By further studying its structure and function, insight could be gained on not only the protein's effect on the lytic cycle, but also its secondary role it may play in the cause of MS.Support or Funding InformationThis project was supported by UW ‐ La Crosse College of Science and Health, UW ‐ La Crosse Faculty Research Grant, and the Hardy Chan and Sons Undergraduate Research Fellowship.
Engineering Hsp104 Variants to Counter Protein Misfolding
Authors: Jackrel Meredith , Shorter James 
Published: November 2023

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP‐43, FUS, and α‐synuclein proteotoxicity by mutating single residues in the middle domain or nucleotide‐binding domain 1 of Hsp104. Strikingly, we have uncovered numerous missense mutations throughout these regions that potentiate Hsp104. Hsp104 potentiation stems from loss of amino acid identity rather than introduction of specific interactions. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease‐associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.Support or Funding InformationTarget ALS Springboard Fellowship
A Common Mechanism of Proteasome Impairment by Neurodegenerative Disease‐Associated Oligomers
Authors: Thibaudeau Tiffany Ann , Anderson Raymond , Smith David M 
Published: November 2023

Neurodegenerative diseases are characterized by an accumulation of misfolded proteins, loss of proteostasis, and the progressive death of neurons. It is well established that the ubiquitin proteasome system (UPS), a major regulator of proteostasis, is impaired in essentially all neurodegenerative diseases. Accordingly, targeted proteasome inhibition in the brain recapitulates many physiological and behavioral hallmarks of neurodegeneration. Several studies have attempted to investigate how the UPS is impaired by protein aggregation, but none of these models provide detailed mechanisms of impairment nor can they be applied to the general spectrum of neurodegenerative diseases. Here, we show that three proteins implicated in Alzheimer's, Parkinson's, and Huntington's diseases form soluble oligomers sharing a similar structure that potently inhibits the proteasome with low nanomolar affinity. These oligomers bind to the 20S proteasome core particle and prevent opening of the substrate‐entry channel, thereby impairing both ubiquitin dependent and independent protein degradation. Mechanistic analysis demonstrates that these oligomers allosterically block a well characterized conformational change in the α‐subunits that is required for substrate entry into the catalytic chamber of the 20S proteasome. Proteasome impairment by these oligomers is expected to accelerate further protein accumulation in a positive feedback loop, thereby contributing to the onset and progression of disease. Our findings suggest that proteasome impairment in many neurodegenerative diseases may have a common mechanism, and identify an excellent target for future drug discovery efforts.Support or Funding InformationThis work was supported by start‐up funds to DMS from West Virginia University and by NIH‐R01GM107129 to DMS.
Evaluating the Catalytic Role of a Conserved Glutamate Residue in Triosephosphate Isomerase from Trypanosoma Brucei Brucei
Authors: Khoury Chris B. , Seangmany Nessa , Chang Timothy C. , Schwans Jason P. 
Published: November 2023

It is well known that enzymes differ from small‐molecule catalysts by use of non‐covalent interactions to position active site residues, but our understanding of the relative importance of residues in this positioning is limited. In triosephosphate isomerase (TIM), a conserved residue, Glu97, has been suggested to be important for catalysis and may be important for positioning a key active site lysine residue (Lys13). However, the Glu97Asp mutation in TIM from Gallus gallus was reported to have no effect on activity, whereas the Glu97Asp mutation in TIM from Plasmodium falciparium led to a 100‐fold rate decrease. To evaluate the catalytic importance and role of Glu97 in TIM and compare to previous results in other organisms, we mutated the Glu97 to aspartate and glutamine in TIM from Trypoansoma brucei brucei (TBB). We determined the effect of these mutations on activity and structure using kinetics, circular dichroism (CD), and fluorescence experiments. Mutations of Glu97 to Gln and Asp led to approximately 24 and 18‐fold rate decreases, respectively. While the mutations affected enzyme activity, the rate effects were lower than the 100 and 4000‐fold rate decrease reported for Glu97Asp and Glu97Gln, respectively, in TIM from Plasmodium falciparium. To evaluate the effects of the mutations on secondary and tertiary structure, CD and fluorescence experiments were performed. CD and fluorescence spectra for the mutants were similar to the wild type; protein denaturation experiments with guanidine hydrochloride showed that denaturation profiles for the mutants were similar to those of wild‐type TIM. This suggests that although Glu97 is important for TBB TIM catalysis, the mutations do not have a significant effect on enzyme structure. We are currently using double mutant cycle analysis with Glu97 and active site mutations to further investigate the importance of Glu97 in positioning active site residues.Support or Funding InformationThis work was supported by a Cottrell College Science Award from the Research Corporation for Science Advancement.
Delving into the details: Investigation of the kinetic properties of SpeG Spermidine N‐acetyltransferases from Escherichia coli and Vibrio cholerae
Authors: Kuhn Misty L , Dang Joseph , Hong Winnie , Jung Ellison , Tran David , Asaro Danielle , Jew Kristen 
Published: November 2023

Biofilms are communities of bacteria found in a variety of environments, including the intestinal mucosa of humans and on surfaces of medical devices. These biofilms protect bacteria from antibiotics and other adverse environmental stresses. Polyamines are polycationic small molecules that are involved in biofilm formation. When polyamines are acetylated, biofilm formation increases. One enzyme that acetylates polyamines in bacteria is the spermidine N‐acetyltransferase SpeG. This enzyme is important for regulating polyamine levels and responding to bacterial stress conditions, but its role in biofilm formation is still unknown. We previously determined the structures of the SpeG enzymes from Vibrio cholerae (VcSpeG) and Escherichia coli (EcSpeG), and found they exist as dodecamers and posses an allosteric site where polyamines bind. This site is not found in the human spermine/spermidine acetyltransferase (SSAT) enzyme, but they share a common core Gcn5‐related N‐acetyltransferase scaffold. These structural differences provide a unique opportunity to study the effect of the allosteric site on enzyme activity and regulation of polyamine levels in different organisms. As a first step toward elucidating the functional properties of SpeG, we present the kinetic characterization and comparison of VcSpeG and EcSpeG enzymes and show how they exhibit distinct properties between the two organisms and the human SSAT enzyme. Additionally, we present a structural comparison of the three enzymes to provide insight into their kinetic differences. Understanding the kinetic details of SpeG polyamine acetylation will ultimately provide important insight for designing drugs that specifically target SpeG over the human SSAT enzyme to prevent or reduce biofilm formation and pathogenesis.Support or Funding InformationStartup funds to MLK from San Francisco State University
Identification of Tumor Necrosis Factor‐α Inhibitors from the Traditionally Used Chinese Plant Mappianthus iodoides
Authors: Garrison Meagan , Park Hyo , Wright Matt , Farone Anthony 
Published: November 2023

The inflammatory response is designed to protect the human body from foreign pathogens, including bacteria, fungi, virus, and parasites. However, when inflammation is uncontrolled it can lead to serious inflammatory and autoimmune diseases. The tumor necrosis factor‐α (TNF‐α) inhibiting drugs currently used as treatment for these diseases, primarily non‐steroidal anti‐inflammatory drugs (NSAIDs), have severe side effects; therefore, there is a desperate need for less toxic, alternative treatments. Traditional Chinese Medicine has grown in popularity in the United States over recent years and many plants are being studied for their medicinal purposes. In the present study, 12 fractions of one Chinese plant, Mappianthus iodoides, were screened for cytotoxicity to human macrophage‐like cells using an Alamar Blue cell viability assay and the non‐toxic concentrations were then assayed for TNF‐α inhibitory activity using enzyme‐linked immunosorbent assay (ELISA). Two fractions exhibited >90% inhibition of TNF‐α at 50 μg/mL, four fractions showed inhibition at 25 μg/mL, and two fractions inhibit at 10 μg/mL and 5 μg/mL. Fraction 7 exhibited greater than 90% inhibition of TNF‐α at 5 μg/mL and is being further fractionated into five isolated compounds using High Performance Liquid Chromatography (HPLC). The five isolated compounds will be characterized and identified using Mass Spectrometry and then assayed for cytotoxicity and TNF‐α inhibition to determine whether the pure compounds inhibit inflammation and may therefore be a potential alternative drug.Support or Funding InformationTennessee Center for Botanical Medicine Research (TCBMR)Guangxi Botanical Garden of Medicinal Plants (GBGMP)
The Effects of Silibinin on Colorectal Cancer Cell Line
Authors: Horita Megan Ann , Ezekiel Uthayashanker 
Published: November 2023

Silymarin, a bioactive extract from Silybum marianum (milk thistle), and Silibinin, the active component of Silymarin, have been used as protective supplements against liver disease and are hypothesized to have anticancer properties. We focused on the effects of Silibinin on colorectal cancer cells because it is the major active component in Silymarin and is known to play a key role in cell cycle arrest and apoptosis in cancer cells. We investigated the anti‐proliferative effects of Silibinin on a colorectal cancer cell line (DLD‐1). Silibinin inhibited colon cancer cell proliferation in a concentration‐dependent manner only at the highest concentration. Silibinin treatment led to inhibition of colon cancer cell proliferation and increased apoptosis. The outcome of this research study shows that silibinin could prevent or eliminate colon cancer initiation and/or progression.Support or Funding InformationMH is supported by the DeNardo Education and Research Foundation Fellowship.
PKA subunit balance plays a key role in lipolysis
Authors: Ji Yul , Lee Jung Hyun , Han Ji Seul , Kong Jinuk , Kim Jae Bum 
Published: November 2023

Protein kinase A (PKA) is a cyclic AMP (cAMP)‐dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. In this study, we have investigated that the stoichiometric balance between catalytic and regulatory subunits is crucial to maintain basal PKA activity and lipid homeostasis. In Caenorhabditis elegans, suppression of PKA via RNAi resulted in severe phenotypes, including shortened lifespan, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti‐lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.Support or Funding InformationThis work was supported by grants from the National Creative Research Initiative Program (2011‐0018312) funded by the Ministry of Education, Science and Technology. Y.J., J.S.H., J.K. were supported by BK21 program.
Insight into the mechanism and structural basis for autoinhibition of PTEN by phosphorylation of its C‐terminal tail
Authors: Dempsey Daniel Robert , Chen Zan , Thomas Stefani , Hayward Dawn , Bolduc David , Cole Philip 
Published: November 2023

PTEN is a tumor suppressor that negatively regulates the PI3K/AKT signaling pathway by functioning as the lipid phosphatase for phosphatidyl inositol 3,4,5‐triphosphate (PIP3). Alterations in the PI3K/AKT pathway have tremendous influence on the oncogenesis of cancer, whereby loss of PTEN function leads to aberrant changes in cell proliferation, growth, survival, and protein synthesis. There are multiple modes of altering the cellular phosphatase activity of PTEN, including decrease or loss of expression, mutation, or post‐translational modification. A cluster of post‐translational phosphorylation sites are located on the C‐terminal tail at residues 380, 382, 383, and 385, which serve to modulate the lipid phosphatase function of PTEN by driving a conformational shift from an open active to closed inhibited state, resulting in a decrease in enzymatic activity and reduction in lipid membrane binding. Many questions centered on the mechanism of PTEN's C‐terminal tail dependent autoinhibition still remain unanswered. In order to provide a detailed mechanism on the molecular features that contribute to its autoinhibitory state, we employed expressed protein ligation (EPL) to install site‐specific and stoichiometric phosphorylation at these residues. We used photo‐crosslinking and designed mutants, which revealed tail interactions between the C‐tail and both the C2 and catalytic domain. These data have enhanced our understanding of the autoinhibitory conformation of PTEN and may pave the way for the rational design of new therapies for the treatment of cancer.Support or Funding InformationNIH 1F32GM120855
Cation‐independent Mannose 6‐Phosphate Receptor Interacts with Several Components of the Plasminogen Activation System
Authors: Bohnsack Richard , Miller James , Twining Sally , Olson Linda , Dahms Nancy 
Published: November 2023

The 300 kDa cation‐independent mannose 6‐phosphate receptor (CI‐MPR) functions in the biogenesis of lysosomes by trafficking newly synthesized lysosomal enzymes with mannose 6‐phosphate (Man‐6‐P)‐containing glycans to the late endosome. In addition, CI‐MPR binds a number of proteins at the cell surface in a glycan‐independent manner, including plasminogen, urokinase‐type plasminogen activator receptor, and insulin‐like growth factor 2. The ability of CI‐MPR to interact with these different ligands is facilitated by its large extracellular region that is composed of 15 contiguous domains. Previous studies have shown that plasminogen binds to CI‐MPR through one or more of its lysine‐binding kringle domains. We report here that tissue‐type plasminogen activator (tPA), which contains two kringle domains, also binds to CI‐MPR. Using surface plasmon resonance, we demonstrate that tPA binds with high affinity to the N‐terminus of CI‐MPR and that CI‐MPR domain 1 appears to be sufficient for binding. The interaction of tPA with CI‐MPR can be partially inhibited by the lysine analog 6‐aminohexanoic acid and by Man‐6‐P, however, the exact nature of the protein interaction is unknown. Addition of CI‐MPR to tPA and plasminogen enhances the rate of plasmin formation. This rate enhancement shows saturable kinetics for receptor with a K½ of approximately 120 nM. The rate of enhancement is dependent on more than one domain of the receptor as CI‐MPR domain 1 by itself is not sufficient to enhance tPA‐dependent plasminogen activation. We propose that CI‐MPR acts as a scaffold to bring tPA and plasminogen together and augments conversion of plasminogen to plasmin by tPA. Angiostatin K1‐4 protein, which includes the pan domain and kringles 1‐4 of plasminogen, is able to inhibit the tPA‐dependent activation of plasminogen in the presence of CI‐MPR. Addition of a lysosomal enzyme also inhibits the CI‐MPR enhancement of plasminogen activation by tPA in a concentration‐dependent manner. Characterization of the mechanisms of these inhibitors will help in understanding the interaction of CI‐MPR with tPA and plasminogen along with providing a better understanding of CI‐MPR's ability to interact with multiple ligands.Support or Funding InformationFunding: NIH R01DK042667