bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Non‐Canonical Function of Leucyl tRNA Synthetase Negatively Regulates Skeletal Myogenesis
Authors: Son Kook , Khanna Nidhi , Banerjee Aditi , Martinis Susan , Chen Jie 
Published: November 2023

Aside from its house‐keeping function in protein synthesis, leucyl tRNA synthetase (LeuRS) has a non‐canonical function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also governs skeletal myogenesis, but its signaling network is distinct from that in cell growth regulation. We set out to investigate a potential role of LeuRS in skeletal myogenesis. Knockdown of LeuRS enhanced myoblast differentiation in vitro, whereas overexpression of LeuRS inhibited myogenesis. In a mouse model of injury‐induced adult muscle regeneration, local knockdown of LeuRS enhanced regeneration. Hence, LeuRS acts as a negative regulator of myogenesis both in vitro and in vivo. However, inhibition of the translational function of LeuRS by leucinol severely impaired myogenesis, consistent with the notion that protein synthesis is required for the myogenic process. These observations suggest a non‐translation role for LeuRS in suppressing myogenesis. Indeed, we found leucine‐binding but not tRNA charging activity of LeuRS to be necessary for its overexpression effect on myogenesis, suggesting that LeuRS may act as a leucine sensor in its function to ensure homeostasis of myogenesis. To probe the mechanism underlying this non‐canonical function of LeuRS, we examined the involvement of Rag GTPases, mediators of LeuRS activation of mTORC1 in cell growth. Constitutively active Rag reversed the effect of LeuRS knockdown on myogenesis and, conversely, Rag knockdown overcame the inhibitory effect of LeuRS overexpression. Rag knockdown also enhanced muscle regeneration in vivo, mirroring the effect of LeuRS knockdown. Taken together, our findings reveal for the first time a function for LeuRS in modulating the homeostasis of myogenesis. This new LeuRS function is mediated by a Rag‐mTORC1 pathway that negatively regulates the myogenic IRS1‐PI3K‐Akt pathway.Support or Funding InformationKeck Foundation, NIH/NIAMS R01AR048914
Cellular protein p32/gC1qR recruits PKC to Viral Protein ICP34.5 and Facilitates HSV Nuclear Egress
Authors: Zhang Cuizhu , Wu Songfang , Wang Yu , Zhang Liming , Pan Shuang , Liu Yaohui , Yang Mengmeng , Chen Da , Wang Jiyan , He Bin , Cao Youjia 
Published: November 2023

The nuclear envelop is considered anatural barrier of virus budding and efficient replication. Several lines of evidence suggest that the disruption of the integrity of nuclear lamina correlate with the nucleocapsid budding of herpesviruses. Mammalian cellular protein p32 is identified as a new target for Herpes simplex virus 1 (HSV‐1)virulent protein, ICP34.5. We found that the efficient nuclear egress of HSV‐1 particles was impaired in the absence of cellular p32 protein. Lacking ICP34.5 encoding gene in HSV‐1 or reduced expression of host p32 severely affects viral replication. Here we report that the interaction betweenp32 and ICP34.5 of HSV‐1 facilitates nucleocapsid release to the cytoplasm. P32binds to the NH2‐terminus of ICP34.5 and recruits PKC to the nuclear lamina, where LaminA/C is phosphorylated and nuclear integrity disturbed. Unlike wildtype virus, an HSV mutant devoid of ICP34.5 or its amino‐terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In HSV infected cells, p32 is recruited to the nuclear membrane and triggers PKC activation whereas in cells infected by HSV with ICP34.5 mutants fail to exert any effect. These results suggest that disintegration of the nuclear lamina mediated by the association of p32 and ICP34.5 promotes HSV replication via PKC.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (31370862; 81672010 to YC)
Generation of TYRO3 Receptor Tyrosine Kinase Clones to Study Interactions with SH2 Domain Proteins in the Retinal Pigment Epithelium
Authors: Harris Lauren , Shelby Shameka 
Published: November 2023

The retina is comprised of cone and rod photoreceptors that must continually be maintained in order to preserve visual acuity. Daily light exposure to the outer portions of the photoreceptors, termed outer segments (OS), leads to photo‐oxidative stress. To combat potential retinal damage caused by light exposure, the Retinal Pigment Epithelium (RPE) phagocytizes spent outer segments. Disruption of OS phagocytosis leads to the accumulation of debris that blocks the flow of oxygen and nutrients to the retina. This will eventually lead to atrophy of the retina and, ultimately, blindness. Previous studies have demonstrated the requirement of Mer Receptor Tyrosine Kinase (MERTK) in the process of OS phagocytosis. Protein interactions between MERTK and SH2 domain proteins Grb2, P85α, Src, and Vav3 have also been shown to be necessary for OS phagoctytosis. Recent studies suggest that TYRO3, a familial receptor tyrosine kinase to MERTK, can compensate in the absence of MERTK. As such, we hypothesized that TYRO3 may bind to SH2 domain proteins known to bind to MERTK. To test the similarities of interactions between MERTK, TYRO3, and associated SH2 domain proteins, various clones of TYRO3 were generated, and GST‐tagged SH2 domain proteins were overexpressed and purified. GST‐SH2 domain proteins Grb2, P85α, Src, Vav1, and Vav3 were overexpressed and purified via affinity chromatography, followed by gel‐filtration chromatography. Two truncated TYRO3 proteins that included the kinase domain and cytoplasmic tail (residues 470 – 809 and 498 – 809) were successfully cloned into a pRSET vector and recombinantly overexpressed. Additionally, full length TYRO3 was successfully cloned into pcDNA3.1 His vector for overexpression in mammalian cultured cells. Further experiments will adequately purify the truncated TYRO3 protein and identify interactions between TYRO3 and SH2 domain proteins recombinantly via Ni‐NTA pull‐down assays. Confirmation of interactions identified in‐vitro will be conducted in mammalian cells transfected with TYRO3. This study has generated the tools necessary to further identify components of the RPE phagocytic mechanism. Elucidation of this mechanism will be instrumental in identifying future retinal disease genes and understanding the impact on proteins that may be involved in Age‐related Macular Degeneration.Support or Funding InformationFlorida Southern College Faculty‐Student Summer Research Collaboration Grant
Toxic PR Poly‐Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers
Authors: Lin Yi 
Published: November 2023

The most prevalent form of familial amyotrophic lateral sclerosis (ALS) involves an expansion of the GGGGCC (G4C2) hexanucleotide repeat located within the first intron of a gene designated C9orf72. While normal individuals typically carry 20–30 G4C2 repeats, affected patients exhibit up to 1,000 or more of the hexanucleotide repeats. Surprisingly, the extended G4C2 repeats can be both transcribed and translated. Translation of either the sense or antisense transcripts of the repeats generates five poly‐dipeptides, GAn, GPn, GRn, PAn and PRn. Two of these polydipeptides, GRn and PRn, show significant cellular toxicity.Missing to date are unbiased studies of the direct intracellular targets of GRn and PRn that may explain the toxicity of these poly‐dipeptides. To address this question, we employed two independent methods, photo‐crosslinking and immunoprecipitation, to identify protein targets of PRn. Both approaches showed that the PRn targets are significantly enriched for proteins bearing low complexity (LC) sequences. By in vitro reconstitution of the interaction between PRn and hnRNP A1, we found that the LC domain of hnRNP A1 is both necessary and sufficient for the interaction with PR20 peptide.When incubated at neutral pH and physiologic ionic strength, the LC domains of numerous RNA binding proteins polymerize into labile, cross‐β fibers. These LC domain polymers then proceed to form hydrogels that mimic the formation of RNA granules. Several independent assays give evidence that PRn binding requires that the LC domains of target proteins exist in a cross‐β polymeric state. First, a series of aliphatic alcohols (1,6‐hexanediol, 2,5‐hexanediol, etc) showed the same hierarchical order of specificity in melting cross‐β fibers, hydrogels as they did in disrupting the interaction between the hnRNPA2 low complexity domain and the PRn poly‐dipeptide. Second, 1,6‐hexanediol, the aliphatic alcohol that has the strongest effect in dissolving cross‐β fibers, was found to impede binding of the PRn poly‐dipeptide to its native, intracellular targets. Finally, a single amino acid substitution (D290V) in hnRNP A2, which strengthens cross‐β polymer formation, also strengthens its binding to the PRn poly‐dipeptide. By constrast, another single amino acid substitution (F291S), which significantly compromises polymer formation, also impedes hnRNPA2 binding to the PRn poly‐dipeptide.In summary, our observations reveal that the intracellular targets of PRn are proteins with low complexity domains and offer mechanistic insight into the toxicity of poly‐dipeptides.
Using Biophysical Characterization to Explore Suppressor of IKKepsilon Structure
Authors: Machek Megan L. , Shikwana Flowreen , Graham Sasha‐Kaye I. , Minzer Ian D. , Wey Ryan , Cruz Richard , Bell Ellis , Bell Jessica K. 
Published: November 2023

The innate immune system is the first line of defense against pathogens. Suppressor of IKKepsilon (SIKE) is a downstream component of the antiviral TLR3 innate immune pathway, although its function in this pathway has not yet been determined. In order to derive information about SIKE function, we undertook characterization of SIKE structure to determine a structure‐function relationship. Using PHYRE2, a SIKE model of the human isoform was predicted that consisted of a V‐shaped coiled coil fold. The secondary structure of SIKE was assessed via circular dichroism. Spectra were consistent with the predicted alpha helical model. Thermal melts (15–80°C) showed a linear, rather than sigmoidal, unfolding transition consistent with computational predictions (GlobPlot, DisEMBL, PONDR), which suggested SIKE contained 35% disordered regions. Addition of up to 25% trifluoroethanol to stabilize helical structure increased the helical content of SIKE by 35%. Fluorescence polarization and size exclusion chromatography assessed the solution size of SIKE (23 kDa) with respect to globular standards. SEC revealed a concentration dependent (0.9 – 3.6 mg/mL) association of SIKE into three distinct species of 79, 185 and 1,325 kDa. Fluorescence polarization of each species compared to standards of lysozyme (14 kDa), albumin (66 kDa) and glutamate dehydrogenase (355 kDa) were consistent with the SEC results. Limited proteolysis coupled to tandem mass spectrometry as well as crosslinking assessed by SDS‐PAGE mapped accessible SIKE sequences and association between SIKE monomers, respectively. Limited proteolysis at 1, 5, and 10 min identified trypsin cleavage sites consistent with a dimeric model of SIKE. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) identified predominantly a dimer species as well as a smaller proportion of a tetrameric species. In the cleft of the V‐shaped coiled coil, 3DLigandSite proposed a zinc binding site. To confirm an interaction between SIKE and divalent cations, SIKE‐ANS titration and fluorescence‐based thermal shift assays were completed +/− Mg, Mn, Ca, and Zn. In each case, a subset of ions perturbed SIKE structure, demonstrating a divalent cation interaction. In conclusion, using various biophysical techniques, we have found that SIKE's structure has regions of disorder bookended by alpha helices; SIKE monomers associate primarily into a dimeric species; divalent metals contribute to SIKE stabilization, and the homology model presented is consistent with these biophysical characterizations.Support or Funding InformationWork supported by NIH grant R21AI107447 and USD SURE program.
Destabilization of the Non‐Polar Subdomain of Hemolysin A Inhibits Hemolysis
Authors: Brunner Madeline Rose , Grilley Daniel , Weaver Todd Michael 
Published: November 2023

Hemolysin A (HpmA), found within Proteus mirabilis, was the focus of this research. HpmA is a member of the two‐partner secretion pathway (TPS), which is the most common mechanism of protein secretion implemented by gram‐negative bacteria. TPS pathways feature both an A and B component, where TpsB secretes and activates its associated TpsA partner across the gram‐negative bacterial outer membrane.Functionally, the 1,557 residue full‐length HpmA protein (fHpmA) acts as a cytolysin and more specifically as a hemolysin. To investigate the hemolytic activity of HpmA, an amino‐terminal truncated version containing only the first 265 residues of the protein was engineered. The truncated form, HpmA265, is secreted by HpmB, its partner, but is incapable of hemolysis. Therefore, HpmA265 harbors a functional TPS domain, but lacks a functional hemolytic domain. Recent equilibrium unfolding studies have dissected HpmA265 into three subdomains: 1) polar core, 2) non‐polar core, and 3) carboxy‐terminal. Each subdomain has an independent ΔGH2Oo′. The objective of this project is to better understand the relationship between subdomain stability, secretion capability, and hemolytic function.This research targeted a series of residues located within the non‐polar core subdomain. The interior of this subdomain, like many globular proteins, contains primarily non‐polar residues. Three residues, Leu188, Leu194, and Ile197 were targeted for mutagenesis based on their localization within the non‐polar core and their proximity to one another. To disrupt the architecture within the non‐polar subdomain, Leu188, Leu197 and Ile197 were replaced with polar residues. Circular dichroism denaturation studies of L194N illustrated an increase in structure loss during the first transition state indicating that the nonpolar core denatured in conjunction with the carboxy‐terminal subdomain. These results are consistent with variant I197H, which also showed an increased m‐value and ΔCD signal showing destabilization of the non‐polar subdomain. A template‐assisted hemolytic assay was used to monitor the impact of the histidine replacement at I197. Past functional results have noted a biphasic process comprised of an initial lag phase followed by a rapid lytic phase. The I197H variant displayed a 8‐fold increase in lag time and a 8‐fold decrease in the maximum rate of hemolysis. Therefore, the template‐assisted function of HpmA265 appears to reside within the non‐polar core subdomain. Results of this research will help us to better understand the two‐partner secretion pathway as related to bacterial virulence.Support or Funding InformationThis work was supported, in whole or in part, by National Science Foundation Grant MCB1050435 (TMW) and a University of Wisconsin‐La Crosse Faculty Research Grant (TMW).
Structural and Functional Characterization of an F17‐Like CUP Adhesin from Uropathogenic E. coli Isolate UTI89
Authors: Klein Roger Davies , Spaulding Caitlin , Hultgren Scott 
Published: November 2023

Adhesion to biotic and abiotic surfaces is an essential step in the infectious cycle of many human pathogens. To this end, both Gram‐positive and Gram‐negative bacteria have developed a variety of strategies to overcome innate clearance mechanisms and persist within the host. The expression and biogenesis of Chaperone‐Usher Pathway (CUP) pili represent one such strategy. In addition to mediating the formation of bacterial biofilms, these proteinaceous extracellular appendages contain, at their tip, a two‐domain adhesin that is composed of an N‐terminal lectin domain and a C‐terminal pilin domain. The structure of the binding pocket within the lectin domain determines the specificity of that pilus for a given epitope. Individual Escherichia coli species encode and express up to 16 distinct CUP operons, each likely mediating a tropism for a specific habitat or environment within the host. While the importance of many of these pili, such as Type 1, P, and others, in human infection has been extensively elucidated, there still exist hundreds of pili across a broad range of bacterial species whose function has yet to be determined. Using a murine model of gastrointestinal infection, we have demonstrated that the F17‐Like (Ucl) pilus is an important virulence factor in the colonization of the gastrointestinal tract by E. coli species. Analysis of the distribution of this pilus type across E. coli clinical isolates reveals an enrichment in uropathogenic E. coli (UPEC) strains that cause recurrent urinary tract infection (rUTI). These findings suggest that the F17‐like pilus allows UPEC to persist in the gastrointestinal habitat before seeding the urinary tract. To probe the molecular mechanism of this attachment, we have obtained an X‐ray crystal structure of the adhesive domain of the F17‐Like pilus tip. Structural and biochemical comparison of this tip to other known bacterial adhesins involved in intestinal colonization indicates that the F17‐like pilus binds an epitope in the gastrointestinal tract that is distinct from both the Fim and F17 molecular receptor.Support or Funding InformationThis work was supported by Washington University MSTP Training Grant #5T32GM007200‐39, NIH/NIDDK R01 #AI048689, and the Department of Molecular Microbiology Sondra Schlesinger Graduate Student Fellowship
Structural and functional insights into σ1 receptor ligand binding
Authors: Schmidt Hayden Riley , Kruse Andrew C 
Published: November 2023

Originally misclassified as an opioid receptor, the σ1 receptor is a single‐pass transmembrane protein with a diverse pharmacological profile. Though its precise biological function has proven elusive, the σ1 receptor is thought to play a role in neurodegenerative diseases such as Parkinson's disease and ALS, as well as neurological pathologies, with the most notable being addiction and depression. A rich repertoire of ligands have emerged for the receptor, including both compounds traditionally associated with other targets, including haloperidol or cocaine, as well as (+) benzomorphans such as (+)‐pentazocine or unique ligands like PD 144418 or ditolylguanidine. Many of these compounds have been classified as agonists and antagonists, but the molecular distinction between the two classifications is not entirely clear. Here, we employ a combination of biochemical, pharmacological, and structural techniques to examine the basis for σ1 receptor ligand binding and attempt to determine the molecular basis of agonism and antagonism for this enigmatic receptor.Support or Funding InformationThis work is supported by the National Science Foundation Graduate Research Fellowship awarded to HRS.
Stress‐triggered self‐association of an enzyme reverses its catalytic activity
Authors: Yoo Haneul , Goyal Rohan , Drummond D. Allan 
Published: November 2023

A recent explosion of studies centered around phase‐separating proteins is revolutionizing our understanding of cell biology. These proteins can reorganize themselves into massive assemblies, which lack the defined stoichiometry of quaternary structures and therefore go beyond our concept of protein quaternary structure, and form membraneless compartments such as stress granules in response to changes in cellular environment. Most studies in this new field have been focused on probing physical properties of membraneless organelles and their constituent proteins. However, equally important questions arise regarding whether and how the biochemical activities of these proteins change upon reorganization and, if so, what regulatory roles they play.We recently identified dozens of proteins that form quinary assemblies in response to heat shock in budding yeast. Consistent with the general idea of quinary assemblies and stress granules as a transient protein inactivation and/or storage mechanism, most of the identified proteins also experience transcriptional repression during heat shock. However, our attention was drawn to one enzyme that forms quinary assemblies but is translationally upregulated during heat shock, hinting that the self‐assembly of this enzyme unlocks its normally concealed enzymatic activity needed during stress. We have successfully reconstituted the in vitro self‐association of this enzyme, diadenosine tetraphosphate (Ap4A) phosphorylase. Using this system, we explore the relationship between the enzymatic activity and the physical transition of the enzyme from soluble monomers to quinary assemblies.Support or Funding InformationMolecular and Cellular Biology Training Grant (T32 GM007183)
NMR Studies of Ubc9 Mutant Identify Structural Basis for SUMO Target Selection
Authors: Placzek William J , Bjornsti Mary‐Ann , Whitaker Robert H , Wright Christine , Onuiri Joshua 
Published: November 2023

BackgroundThe regulated biosynthesis, trafficking, interaction, and degradation of proteins are essential for normal cell growth. SUMO is a conserved ubiquitin‐like protein that is conjugated to a multitude of cellular proteins and mediates these functions. The transient SUMO modification (SUMOylation) of proteins affects protein function, allowing cells to respond to environmental stress without the need for de novo protein synthesis. Not surprisingly, dysregulation of SUMO conjugation has been directly implicated in the pathogenesis of human cancer and in modulating cellular responses to chemotherapeutics. Yet, the underlying determinants of SUMO target specificity.Materials and MethodsSUMO conjugation is accomplished by an ATP‐dependent cascade of enzymes, which includes a single activating enzyme (SAE1/2 heterodimer), SUMO‐1 or SUMO‐2/3, and a single conjugating enzyme (Ubc9). We used yeast genetics, biochemical SUMO conjugation assays, and NMR spectroscopy to define the basis for alterations in substrate selection and SUMO chain formation.ResultsOur studies suggest that the altered substrate specificity induced by the Ubc9P123L mutant enhances cell sensitivity to a wide range of DNA damaging agents. NMR analyses of wild‐type and mutant Ubc9 enzymes have identified critical flexibility in Ubc9 necessary for SUMO target selection. In parallel, biochemical and genetic studies demonstrate the impact this dysregulation of Ubc9 function has on SUMO conjugation and cell viability.ConclusionsOur results provide new insights into the structural features of Ubc9 that underlie substrate selection and the impact of SUMO chain linkages on cell viability. These findings constitute a paradigm shift in our understanding of SUMOylation and the potential contribution of SUMO chain linkages to tumorigenesis and chemotherapeutic drug response.