bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining functional variation of diverse Hsp104 homologues
Authors: March Zachary , Shorter James
Published: November 2023

Hsp104 is a hexametric AAA+ protein disaggregase from yeast that can rapidly disassemble disordered aggregates, preamyloid oligomers, amyloids, and prions. These protein‐remodeling activities enable resistance to and reversal of stress‐triggered protein misfolding, and have even allowed yeast to harness prions for adaptive purposes. Hsp104 orthologues are found in all nonmetazoan eukaryotes and eubacteria, but the vast majority of these remain unexplored. Thus, Hsp104 sequence space, and the extent of protein‐remodeling activities enabled by it, is unknown. Here, I present our efforts to functionally define diverse Hsp104 homologues from fungal, plant, algal, and protozoan lineages. While these homologues functionally complement various protein‐remodeling activities performed by Hsp104 from S. cerevisiae (ScHsp104), we find, surprisingly, that they are able to antagonize unique and diverse proteotoxic misfolding events under conditions where ScHsp104 is inactive. I also present our work to understand the molecular mechanisms underpinning these activities. Our results establish that natural Hsp104 orthologues can have therapeutic‐disaggregase activity against substrates involved in human disease. We suggest that sequence variation among Hsp104 orthologues may be a valuable resource in the engineering of therapeutic disaggregases to counter disease.
Understanding the influence of translation‐elongation kinetics on protein structure and function
Authors: O'Brien Edward Patrick
Published: November 2023

An emerging paradigm is that translation kinetics can influence nascent protein behavior. Introducing synonymous codon mutations into an mRNA molecule, which changes the rate at which codon positions are translated by the ribosome but not the encoded protein's primary structure, has been shown to influence whether a nascent protein will fold and function, misfold and malfunction, aggregate, or efficiently translocate to a different cellular compartment. Synonymous mutations that can change translation rates have now been linked to a variety of diseases, including subtypes of hemophilia and cancer. These findings are a shift away from the thermodynamic‐control paradigm of protein structure and function to one in which kinetic control can play an important role in determining nascent protein behavior. My lab is developing theoretical and computational tools to understand, model and predict the coupling of in vivo protein behavior to translation‐elongation kinetics. I will present results from two of our recent studies in which we demonstrate that by using chemical kinetic modeling we can accurately predict co‐translational folding curves in vivo, and that by using coarse‐grained molecular‐dynamics simulations we can explain the physical origin of the experimental observation of critical‐codon positions. Critical codons are codon positions within a messenger RNA that, upon a synonymous substitution, cause the nascent protein to behave in a radically different manner. The tools we are developing can be applied to address questions concerning translation‐elongation effects on single molecules up to the entire proteome of organisms.
New Insights from High‐throughput Biophysical Screening of Protein‐sequence and Coding‐sequence Libraries
Authors: Allen Benjamin
Published: November 2023

The convergence of next‐generation sequencing, large‐scale gene synthesis, and high‐throughput screening has enabled unprecedented, large‐scale characterization of the sequence and contextual factors that govern protein‐assembly outcomes. We have leveraged existing fluorescent readouts for protein solubility, protein aggregation, and enzyme function to evaluate up to 1 million defined coding‐sequence variants of individual protein scaffolds in living bacterial cells. In one example, we targeted diversity to the hydrophobic core of a small, stable protein domain. Preliminary analysis suggests that there are divergent routes to solubility in this system: both minimally hydrophobic and highly hydrophobic sequences can assemble into soluble forms, while soluble assembly may be more exceptional for sequences with intermediate hydrophobicity like the wild type. In another example, we built a library of synonymous coding sequences with either a common or a rare codon at each degenerate site. While this library showed variation in soluble and total protein production, the observed variation could not be explained by simple models of translation initiation or elongation. Contrary to expectation, many highly expressed constructs contained a large number of low‐abundance codons, suggesting that factors other than tRNA availability may be rate limiting in the production of soluble protein. These findings suggest alternative approaches to the engineering of protein solubility and the optimization of coding sequences for high expression.

Support or Funding Information

The Department of Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, the Pennsylvania State University
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 Information

NSF 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.
Characterization of Recombinant Fructose‐1,6‐Bisphosphatase (FBPase) Gene Mutations: Insights into Modulation of FBPase activity through Gene Mutation
Authors: Topaz Gemma , Epiter‐Smith Victor , Emad Megan , Ford Vanessa , Daley Jadine , Silva Christine , Subramanian Manjula , Sosa Harolin , Stieglitz Kimberly
Published: November 2023

Diabetes is a disease affecting how the body regulates glucose. Multiple factors contribute to Type II Diabetes resulting in hyperglycemia. Previous studies in patients with hypoglycemia identified two mutations in the coding region of the fructose‐1, 6‐bisphosphatase (FBPase) gene. Based on the mutations identified, the initial focus of our project was a full kinetic analysis of these mutations using the recombinant pig kidney FBPase enzyme. Results of this analysis showed an approximately 8‐fold decrease in the relative catalytic constant (Kcat) and catalytic efficiency (CE) compared to wild‐type enzyme (WTE). A separate mutation was done in the active site of the enzyme in order to investigate if the enzyme could be activated. Kinetic analysis of this mutation showed a 30‐fold increase in the relative Kcat and catalytic efficiency (CE). Other mutations in the adenosine monophosphate (AMP) binding‐site (K112A and Y113A) showed marked decrease in AMP inhibition (AMP is the enzyme's natural inhibitor). Kinetic analysis of the enzyme in the absence of AMP resulted in maximum velocity (Vmax) values for K112A and Y113A mutants that were similar to the wild‐type Vmax but which were elevated at higher concentrations of FBP substrate. This suggests that mutations of this type promote hyperglycemia by preventing AMP inhibition of the FBPase in vivo. Clinical implications for these results are discussed in relation to the project.
α‐Effect Furnishes a Mechanistic Bypass for General Base Catalysis in Hedgehog Protein Autoprocessing
Authors: Callahan Brian , Giner Jose L , Ciulla Daniel , Jorgenson Michael
Published: November 2023

Eukaryotic signaling proteins in the “hedgehog” family undergo a rare autoproteolytic maturation reaction involving nucleophilic attack by cholesterol. A recent mutagenesis study indicated that a conserved aspartate residue (D303) of hedgehog functions as an essential general base, activating the 3‐OH group of cholesterol [Xie J, Owen T, Xia K, Callahan B, Wang C. (2016) J Am Chem Soc. 138(34):10806–9.]. Accordingly, autoprocessing of a point mutant (D303A) was reduced by a factor of >104 fold. Here we report that this ostensibly dead mutant can catalyze autoprocessing at near wild‐type levels when cholesterol is replaced by a synthetic sterol, 3‐hydroperoxycholestane (3‐HPC), in which the sterol ‐OH is substituted with ‐ OOH. Other hedgehog point mutants at D303 also accepted 3‐HPC as a substrate albeit to varying degrees, with the rank order of activity: D303H>D303A≈D303N>D303R>>D303E. We attribute the remarkable substrate rescue by 3‐HPC to the alpha‐effect, where nucleophilic groups with tandem electronegative atoms exhibit anomalously high reactivity despite relatively low basicity.
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.
The E. coli and Human Nudix hydrolases NudC and NUDT12 cleave damaged NADH
Authors: Ray Atreyei , Beaupre Brett A. , Moran Graham R. , Frick David N.
Published: November 2023

Nudix enzymes hydrolyze phosphoanhydride bonds linked to nucleotides. One subclass of Nudix enzymes cleaves dinucleotides to yield two nucleoside monophosphates. The prototype enzyme of this Nudix subclass is the product of the E. coli nudC gene (aka NudC or Orf257), which has been previously shown to cleave NAD(P)H, NAD(P)+, ADP‐ribose, AppA, and NAD‐capped RNA. Since other Nudix enzymes are known to remove potential mutagens from nucleotide pools, we tested the hypothesis that NudC‐like proteins might cleave potentially harmful dinucleotides. Recent studies have shown that many cells express enzymes that cleanse cells of a form of β‐NADH that carries the hydride in the 6‐position of the nicotinamide base (6DHNAD). 6DHNAD is a potent inhibitor of numerous cellular oxidoreductases. In this study, NudC and its human homolog NUDT12 were expressed in E. coli, purified, and their abilities to cleave 6DHNAD were analyzed. The enzymatic activities of each enzyme were quantified using a colorimetric assay and High Pressure Liquid Chromatography. Both proteins required divalent metal ions to hydrolyze 6DHNAD to yield two nucleoside 5′ monophosphates.
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 Information

This 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 Information

Department 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 Information

This 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 Information

This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01.
Unmixing enzyme allostery
Authors: Meisburger Steve , Ando Nozomi , Taylor Alexandra B. , Kahn Crystal A. , Zhang Shengnan , Fitzpatrick Paul F.
Published: November 2023

Protein allostery is a key mechanism of metabolic control in all forms of life. Enzymes that change their activity in response to an effector are particularly important for regulating metabolite fluxes. I will discuss the application of small‐angle X‐ray scattering (SAXS) to study allosteric enzymes, which exist as complex mixtures in solution, using new strategies to “computationally purify” complex mixtures. In particular, I describe the powerful combination of chromatography‐coupled SAXS and evolving factor analysis (EFA), an extension of singular value decomposition (SVD), which can separate scattering profiles of species that are not completely resolved by the column. We have used this technique to visualize the domain motions of phenylalanine hydroxylase (PheH), an enzyme that converts phenylalanine to tyrosine in the liver. The data support a model where tetrameric PheH is activated by dimerization of the regulatory domains, which bind phenylalanine in the active conformation. Surprisingly, the allosteric mechanism of this mammalian enzyme appears to be conserved from bacterial enzymes that synthesize aromatic amino acids.
A Novel, Green Synthesis of Deuterium Labeled Compounds
Authors: Kadish Dora , Kokel Anne , Torok Marianna , Torok Bela
Published: November 2023

While deuterium (D) is barely different from its most abundant isotope hydrogen (H), the additional neutron makes a significant difference in the properties of deuterated compounds. While metabolic enzymes easily transform drug molecules to metabolites that the body can excrete, the introduction of deuterium to drugs appears to strengthen the resistance of drugs toward metabolism. The higher the stability, the longer the drug can work, which may result in lower dosage, therefore less side effects. In fact, the carbon‐deuterium bond is known to be six‐ten times stronger than its C‐H counterpart. There are several known methods for the introduction of deuterium to organic compounds; most methods, however, do not conform to the recent expectations and standards of sustainable synthesis. In order to develop an environmentally benign deuteration method we have turned our attention to the Al‐H2O system that is commonly applied for hydrogenation reactions. Replacing the H2O with its deuterated version D2O this hydrogen generating system can be turned to an easy and safe source of deuterium. The application of either the commercially available Ni‐Al alloy or Al only in conjunction with common hydrogenation catalysts, such as Pd or Pt, for the H‐D exchange of compounds with reactive C‐H bonds can be easily performed, while yielding no harmful byproducts. The low reactivity of the aluminum metal can be significantly enhanced by the application of ultrasonic irradiation. The H‐D exchange reaction can be carried out under relatively mild conditions; in more difficult cases the application of microwave irradiation yields the final products in a 1 h reaction. The success of the method was demonstrated by applying a broad variety of compounds from essential amino acids to actual drug compounds. By generating stronger bonds, the drugs would be able to remain active for an extended period of time, allowing the use of lower amounts, thus significantly improve the safety of their use.
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 Information

National Institute of Health GM099904‐04
Mechanistic comparison of structurally divergent transcriptional coactivators through covalent activator‐coactivator complexes
Authors: Henderson Andrew R , Beyersdorf Matthew , Foster Nicholas , Sanford Kevon , Henley Matthew , Mapp Anna
Published: November 2023

Transcriptional coactivators act as essential linchpins between DNA‐bound transcriptional activators and the rest of the transcriptional machinery. The coactivator Med25, of the human Mediator complex, contains a unique coactivation domain, the Activator Interacting Domain (AcID), which links several disease‐associated activators and the transcriptional machinery. The AcID motif is composed of both alpha helices and beta sheets whereas the most well characterized coactivation domains (KIX and TAZ1) are entirely alpha helical in structure. The molecular basis of activator‐coactivator interactions are most thoroughly understood in those between activators and the KIX domain of CBP/p300. Intrinsic disorder and allostery have been identified as critical KIX features for mediating binary and ternary complex formation between transcriptional activators and KIX at two distinct activator binding sites. We sought to probe the molecular basis of AcID‐activator interactions specifically examining the roles of intrinsic disorder and allostery in mediating AcID‐transcriptional activator complexes. The transcriptional activator, VP16 (Herpes simplex virus), contains two tandem, yet functionally independent, transcriptional activation domains (TAD) that have been shown to interact with AcID at two distinct binding surfaces. These protein‐protein interactions (PPI) were probed via the construction of cysteine‐containing VP16‐derived peptides that can be covalently tethered to AcID through the formation of activator‐AcID disulfides. Native, or mutationally introduced, cysteines at several positions on AcID enable specific formation of VP16‐AcID binary complexes at either AcID binding surface. This selective control enables us to probe activator binding at each binding surface individually, vastly simplifying our analysis of these binding events and allowing observation of essential differences in the mechanism of binary and ternary complex formation of the AcID and KIX domains. A suite of direct binding, kinetics, and NMR performed using these covalent complexes have revealed that Med25 AcID, with its unique beta‐sheet rich architecture, diverges from KIX in its mechanism of transcriptional activator complex formation. AcID, like KIX, possesses an allosteric network linking both activator binding surfaces but these AcID binding sites differ in their ability to accommodate substrates bound in many different conformations forming so‐called “fuzzy” interfaces. This refined understanding of Med25 AcID has guided efforts to develop small‐molecule inhibitors of AcID capable of inhibiting specific activator‐AcID interactions.
PTEN Regulation by WWP2
Authors: Chen Zan , Dempsey Daniel , Xu Wei , Li Xiaoguang , Devreotes Peter , Wolberger Cynthia , Gabelli Sandra , Cole Philip
Published: November 2023

WWP2 is a member of NEDD4 family HECT E3 ubiquitin ligases. The architecture of the NEDD4 family proteins include an N‐terminal C2 domain, two to four WW domains, and a C‐terminal HECT catalytic domain. The NEDD4 family E3 ligases play important regulatory roles in development and differentiation, immunological function, and cancer development, and their activity needs to be tightly regulated to prevent pathogenesis. WWP2 is the E3 ligase that catalyzes Lys ubiquitination of numerous proteins including key transcription factors such as OCT4 and signaling molecules such as PTEN. Our lab has been interested in studying how PTEN C‐tail phosphorylation (residues Ser380, Thr382, Thr383, and Ser385) regulates its ubiquitination by WWP2. Using expressed protein ligation, we generated semisynthetic non‐phosphorylated and tetra‐phosphorylated PTEN, and found that the non‐phosphorylated PTEN is more efficiently ubiquitinated by WWP2 than the phosphorylated PTEN, indicating that C‐tail phosphorylation of PTEN could inhibit its ubiquitination by WWP2. In the course of our studies, we have found that WWP2 itself is regulated by autoinhibition. We have dissected aspects of WWP2 that contribute to its autoinhibition and will discuss how the protein may be regulated to ensure proper catalytic tone.
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 Information

This work is supported by National Institutes of Health Grant R01 GM107175 and R42 DA037622
Determining the Correlation between Drosophila melanogaster Toluene Exposure and the Resulting Toxicity Effects on Fly Survival and Fecundity
Authors: Trivino Vanessa , MacGregor Michael , Nguyen Tang , Nunez Brenda , Lodhra Zara , Castillo Laura , Luu Brenda , Rosell Rosemarie , Ledesma Elmer
Published: November 2023

Toluene is a volatile hydrocarbon found in industrial settings and in household products. It is classified as noncancerous but air‐toxic, and chronic exposure to toluene can have negative effects on human neurological function, organ function, fetal development, and can cause hormonal changes. Our purpose is to investigate the effects of toluene on a population of the model organism, Drosophila melanogaster. Since humans and D. melanogaster have similar nervous system structure and function, and exposure to toluene results in permanent damage to the central nervous system, we anticipate that D. melanogaster will show similar results. In fruit flies, toluene is known to negatively affect fly eclosure and delay pupation patterns. To observe these effects, a continuous flow system that delivers known concentrations of gaseous toluene to populations of D. melanogaster was developed. We have interpolated the lethal concentration of gaseous toluene that kills 50% of the population (LC50) to be 279 ppm. Adults exposed at concentrations in the LC50 range were mated and the resulting offspring were counted at egg, larval, and pupal stages. These data allow us to track fecundity and progression of fly development under the influence of parental toluene exposure. In addition, light microscopy is used to observe the effects of toluene exposure on the offspring larvae's morphology, which is compared to the morphology of the larvae from unexposed parents. Interpreting the data will allow us to identify the developmental stage(s) at which fruit flies are affected by toluene exposure.
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.
The PepSAVI‐MS Pipeline for Natural Product Bioactive Peptide Discovery
Authors: Kirkpatrick Christine L , Pritchard David , Parsley Nicole , Liu Yufeng , Hoskin David W , Shaw Lindsey N , Hicks Leslie M
Published: November 2023

The recent increase in multidrug‐resistant pathogens and associated morbidity/mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. While natural products are a well‐studied source of biologically active small molecules, peptidyl factors contributing to their medicinal properties remain largely unexplored. To this end, we have developed the PepSAVI‐MS (Statistically‐guided bioactive peptides prioritized via mass spectrometry) pipeline to identify bioactive peptide targets from complex biological samples. To validate this pipeline, we have demonstrated successful detection and identification of a known antimicrobial peptide, cycloviolacin O2 (cyO2), from the botanical species Viola odorata. Additionally, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cyO2 against E. faecium and A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast and prostate cancer cell lines. The developed platform is highly versatile as it is adaptable to any natural product source of peptides and can test against diverse physiological targets, including bacteria, fungi, viruses, protozoans, and cancer cells for which there is a developed bioassay. As such, we demonstrate extension of the pipeline to bacterial and fungal‐sourced AMPs through identification of the killer toxin KP4 from Ustilago maydis and the bacteriocin Bac‐21 from Enterococcus faecalis harboring pPD1. Using the validated pipeline, we begin to probe the vast array of natural product sources to prioritize highly active species for downstream analysis.
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 Information

The authors wish to thank the NAMS and Provost Travel Funds and the Provost Opportunity Funds.
Anti‐Cancer Properties of the Antimicrobial Peptide CDT on A549 Lung Cancer Cells
Authors: Hendrickson Nathan , Eitel Anna , Jujjavarapu Murali , Guthrie Jeffrey , Evans Hedeel , Heyl‐Clegg Deborah
Published: November 2023

Antimicrobial peptides, showing therapeutic effects against bacteria and cancer, have positive charges which interact with negatively charged membrane components of these cells. In this study, varying concentrations of cysteine‐deleted tachyplasin (CDT) were examined to determine its anticancer effects. CDT was added to human lung cancer cells and incubated in a 96 well plate for several days. In order to measure the number of live cells remaining after treatment, MTT assay was used to measure absorbance. Preliminary results indicate that CDT reduces absorbance, suggesting that it causes tumor cell death.
Disruption of the dopamine D1/D2 heteromer using synthetic peptides
Authors: Champion Margaret , Baraka Adam , Evans Hedeel , Heyl‐Clegg Deborah
Published: November 2023

Dopamine receptor subtypes D1 and D2 interact in a manner that modulates a number of signaling pathways. This interaction is not well characterized and is thought to involve heterooligomerization via interaction of amino acids in the cytoplasmic regions of each receptor. The interaction is presumed to occur between two adjacent arginine resides in intracellular loop 3 of the D2 receptor and two adjacent glutamic acid residues in the carboxyl tail of the D1 receptor. Controversy remains, however, over the validity and nature of this interaction, particularly with regards to formation of a functional D1–D2 heteromer. As evidence exists for the role of the D1–D2 heteromer in the development of major depressive disorder, characterization of the D1–D2 heteromer may lead to novel treatments of this disorder, which affects some 350 million people worldwide. Using purified proteins, PC12 cell extracts and protein lysate isolated from frontal and temporal lobe cells of depressed and non‐depressed patients, we report evidence of D1–D2 heteromer formation, as well as its disruption by a novel sequence of synthetic peptides, which may exert this effect by competing for a binding site between the two receptors. These peptides may present a potential target for drug development in the long‐run.
Characterizing the Impact of the Highly Endosmolytic Cell‐Penetrating Peptide, dfTAT, on Human Cells
Authors: Kondow Helena
Published: November 2023

One of the biggest challenges of delivering molecules into live cells is maintaining cellular homeostasis after delivery. In many reported delivery systems, cell viability decreases as a function of increased delivery efficiency. In our lab, we have generated a highly endosmolytic cell‐penetrating peptide, dfTAT, that aids the delivery of macromolecules into live cells without impacting cell viability or transcriptome regulation. dfTAT enters the cell by endocytosis, and is subsequently released from late endosomes to enter the cytosol. With its highly efficient endosomal release, dfTAT would be expected to be toxic to cells as a result of the predicted release of calcium and hydrolytic enzymes into the cytosol. However, because dfTAT‐mediated delivery does not elicit the same toxic responses as other delivery systems, it is of interest to investigate what is happening to the cell post‐release. One hypothesis is that other cellular processes mask certain physiological changes occurring to the cell. These processes may include repair of damaged late endosomal membranes or autophagy‐dependent degradation of damaged vesicles. Preliminary experiments have indicated that dfTAT‐mediated late endosomal disruption triggers a galectin‐3 response, a hallmark signal of autophagy induction. Based on this initial finding, it is of interest to identify what these cellular response mechanisms are, and what additional proteins may be involved in these processes. Another important aspect of characterizing a cell's response to dfTAT is to quantify how many endosomes are experiencing leakage, as well as elucidating the kinetics of how damaged endosomes are processed by the cell. Overall, the relevance of this work goes beyond intracellular delivery, in that the highly endosmolytic efficiency of dfTAT can be exploited to learn about membrane damage that occurs in other biological systems. In addition, further elucidating the proteomic profile, as well as kinetics, of a cell's response to endosomal membrane damage can be applied to understanding the fundamental biology of a cell.
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 Information

This 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