bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Sodium Hydrogen Exchanger 1 Palmitoylation is Associated with Suppression of Cell Migration
Authors: Hovde Moriah J , Kooiker Amanda J , Rastedt Danielle E , Provost Joseph J , Vaughan Roxanne A , Wallert Mark A , Foster James D 
Published: November 2023

The sodium hydrogen exchanger isoform 1 (NHE1) is involved in many cellular processes including regulation of intracellular pH, coordination of cell migration, control of cell volume, and anchoring protein interactions at the cell membrane. NHE1 is integral plasma membrane protein with 12 transmembrane spanning domains that exchanges an intracellular proton for an extracellular sodium ion. NHE1 is implicated in neoplastic transformation leading to tumorgenesis where the exchanger localizes to the leading edge of migrating cells initiating forward movement of cells indicating a potential role for NHE1 in cancer progression. NHE1 is regulated by many factors including multiple protein‐protein interactions and a number of phosphorylation events. Recent reports indicate that many proteins identified to play key roles in cancer are regulated by palmitoylation. S‐palmitoylation is a dynamic and reversible posttranslational modification, in which the protein undergoes cycles of palmitoylation and depalmitoylation, catalyzed by palmitoyl acyltransferases (PATs) and acyl protein thioesterases (APTs), respectively. Palmitoylation is known to regulate protein trafficking, membrane microlocalization and protein‐protein interactions. These findings prompted us to investigate the presence of this modification on NHE1 using acyl‐biotinyl exchange (ABE), an in vitro assay for post‐hoc detection of endogenous palmitoylation, which revealed that NHE1 is palmitoylated. To further evaluate the nature of NHE1 palmitoylation we treated Chinese hamster lung fibroblasts expressing human NHE1 (PSN) with the irreversible PAT inhibitor 2‐bromopalmitate (2BP). We found 2BP suppresses NHE1 palmitoylation in both a time and dose dependent manner. Additionally, PSN cell migration assessed using electric cell‐substrate impedance sensing was inhibited by 2BP in a manner consistent with the degree of inhibition of NHE1 palmitoylation. These results suggest that palmitoylation may regulate NHE1 function and its role in modulating cell migration.Support or Funding InformationNational Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)
Protein Succination‐A Biomarker of Mitochondrial Stress in Adipose Tissue Inhibits Protein Function and is Regulated by Nutrient Balance
Authors: Manuel Allison Marie , Walla Michael , Piroli Gerardo , Frizzell Norma 
Published: November 2023

S‐(2‐succino)cysteine (2SC) is an irreversible post‐translational modification formed by the reaction of fumarate with protein cysteines, also known as protein succination. 2SC is a biomarker of glucotoxicity driven mitochondrial stress in the adipocyte. Here, we investigated if 2SC is a mechanistic mediator of adipocyte stress via modification of the critical chaperone protein disulfide isomerase (PDI) and examined if 2SC levels could be regulated by caloric restriction.Fumarase knockdown or control 3T3‐L1 adipocytes were matured in normal (5 mM) or high (30 mM) glucose for 8 days, or in 30 mM glucose for 4 days and then switched to 5 mM glucose for 4 days. Adipocyte lysates and adipose tissue protein from control and diabetic mice were analyzed by immunoblotting with an anti‐2SC antibody. The fluorescent PDI specific substrate DiEosinGSSG was utilized to assess PDI enzymatic activity in adipocytes. LC‐MS/MS mass spectrometry was utilized to identify the specific site of recombinant PDI succination.2SC significantly increases in adipocytes matured in high glucose and in the adipose tissue of diabetic mice. Succination significantly lowers PDI reductase activity in fumarase knockdown adipocytes and in the adipose tissue of diabetic mice versus control. MS/MS analysis identified an important PDI active site cysteine as a target of succination. 2SC is turned over in adipocytes when maturation medium is returned to normal glucose.2SC inhibits PDI reductase activity facilitating adipocyte stress as PDI is no longer able to re‐fold proteins within the adipocyte. The turnover of succinated proteins when nutrient balance is restored illustrates the importance of a balanced diet in preventing obesity and diabetes‐related dysfunction in adipose tissue. Our data demonstrates a novel biochemical mechanism mediating nutrient induced stress in the adipocyte, and that this stress is ameliorated when reasonable caloric restriction is employed, ensuring healthy functional adipose tissue.Support or Funding InformationNational Institute of Health 1F31DK108559‐01(AMM), 1R56DK105087‐01(NF) and 1‐11‐JF‐13 from the American Diabetes Association (NF).
Novel Physiological Targets of Fic‐mediated Adenylylation/AMPylation
Authors: Mattoo Seema , Sanyal Anwesha 
Published: November 2023

Post‐translational modification (PTM) of proteins is a common theme in signal transduction. We previously discovered a family of enzymes, defined by a ‘Fic’ domain, that covalently attach an adenosine monophosphate (AMP) to their targets to alter cellular signaling. This PTM, called Fic‐mediated adenylylation/AMPylation is a strategy used by some bacterial pathogens to adenylylate and inactivate host Rho GTPases, thus evading host defense mechanisms. Recently we demonstrated that the sole human Fic, HYPE, adenylylates the ER molecular chaperone, BiP, thereby functioning as a key regulator of ER homeostasis. Accordingly, we showed that HYPE localizes to the ER lumen to modify BiP and alter the unfolded protein response (UPR) to stress. Using electron tomography and biochemical analyses, here we report that HYPE localizes to cellular compartments other than the ER, and have identified new physiologically relevant protein targets for HYPE. Validation of these targets is discussed.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Core Pilot Grant, 2) a Showalter Research Trust Award, 3) an American Cancer Society (ACS) Institutional Grant, and 4) the National Institute Of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.
Unique Solutions to a Common Problem: Understanding How I‐CreI Homologs Recognize the Same DNA Target Sequence
Authors: Polkinghorn Caley Ray , Kaiser Brett , Wylie Nathan , Tang Malu , Ruff Ray 
Published: November 2023

Homing endonucleases (or “meganucleases”) are DNA cleaving enzymes that are widely present in microbial genomes. They are known as “selfish elements” for their ability to cleave their DNA target site within related species and insert themselves into the genome via homologous recombination. We studied a family of related LAGLIDADG homing endonucleases (“I‐Cre1 homologs”) which have been identified within the large ribosomal subunit gene of the chloroplast genome of numerous unicellular green algae. Despite significant divergence at the amino acid level (40–60% identity), these enzymes have retained the ability to cleave nearly identical DNA targets. For this reason, this enzyme family is a great case study to understand protein‐DNA interactions. Our goal was to investigate the unique ways in which a subset of I‐Cre1 homologs cleave their DNA targets. We used X‐ray crystallography to solve the molecular structures of three homologs bound to DNA then constructed contact maps for all of the individual protein‐DNA contacts being made. We also generated cleavage specificity profiles by performing DNA cleavage assays using one‐off mutated substrates for four enzymes (Col2593c, Sob2593c, Cre2593c and Hla2593c). The specificity profiles and DNA‐protein contact maps for the enzymes were analyzed alongside a multiple sequence alignment to identify how key contacts confer regions of specificity and flexibility. These conclusions allow for a detailed analysis of the unique ways each protein recognizes and cleaves their common DNA target.Support or Funding InformationMurdock Charitable Trust (research)
SAXS: A Versatile Tool to Study Biological Macromolecules in Solution
Authors: Ma Weikang , Chakravarthy Srinivas , Shang Weifeng , Irving Thomas 
Published: November 2023

The Biophysics Collaborative Access Team (BioCAT), which operates the high‐brilliance undulator Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is dedicated to studying disordered macromolecules in solution using Small‐Angle X‐ray Scattering (SAXS). In SAXS, the solution scattering profile contains three dimensional information of macromolecules at nanometer scale in solution. The combination of a highly brilliant X‐ray source and advanced micro‐focusing optics at BioCAT enables significant insights into the structure and dynamic of bio‐macromolecules from both static and time‐resolved SAXS experiment.The quality and interpretability of SAXS data is largely contingent on sample homogeneity. Size‐exclusion chromatography (SEC)‐SAXS, which was pioneered at BioCAT has therefore emerged as a powerful mode of data acquisition as it ensures optimal sample quality by purification immediately before exposure to x‐rays. In addition, we have also incorporated multi‐angle light scattering (MALS), dynamic light scattering (DLS), and differential refractive index (dRI) detectors in line with the SAXS camera thus enabling acquisition of a full complement of biophysical data typically required to structurally characterize biological macromolecules simultaneously with SAXS data.The new frontier in the field is to study real‐time structural changes in biological systems as a response to very specific environmental stimuli such as a change in denaturant concentration, ionic strength, pH or ligand binding. Forefront time resolved SAXS techniques using chaotic mixing continuous flow, continuous laminar flow and stopped flow mixing are now available to a wide variety of bio‐medical projects at BioCAT primarily on account of being optimized over the past few years both for sample consumption and mixing efficiency.Support or Funding Information9 P41 GM103622
Exploring the Structural Impact of Unnatural Amino Acids on Protein Structure
Authors: Savidge Nicole S , Maurici Nicole , Brewer Scott H , Phillips‐Piro Christine M 
Published: November 2023

Unnatural amino acids (UAAs) have the potential to serve as effective, site‐specific reporters of local protein structure and dynamics. However, in order to act as an effective probe, the UAA needs to be minimally perturbative to the native protein structure. One potential IR probe of local environment is 4‐nitro‐L‐phenylalanine (pNO2F). pNO2F has been genetically incorporated into the model protein superfolder green fluorescent protein (sfGFP) at a solvated and partially buried site individually in order to determine the structural impact of the presence of the UAAs on the native protein structure. The IR analysis of these protein constructs in addition to the protein crystal structures will be presented.Support or Funding Information• The F&M Hackman Scholars Program and Schappell Research Fund• NIH R15 (2R15GM09330) to S.H.B/E.E.F.
Significance of charged residues in the catalytic sites of Escherichia coli ATP synthase
Authors: Ahmad Zulfiqar 
Published: November 2023

BackgroundF1Fo ATP synthase, the smallest biological nanomotor, is the fundamental means of cellular energy production in almost all living systems. Oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts generates ATP from ADP and Pi (inorganic phosphate). The molecular mechanism of ATP synthase and generation of ATP depends on the designated catalytic site residues. We have been interested in identifying the specific Pi binding residues in the cyclical process of binding change mechanism. So far we have investigated eleven residues, βArg‐246, βAsn‐243, αArg‐376, βLys‐155, βArg‐182, αPhe‐291, αSer‐347, αThr‐349, αIle‐346, αIle‐348, and αGly‐351 based their proximity to the phosphate analogs AlF3 or SO42− in x‐ray structures of catalytic sites. We suspect that many other catalytic site residues including highly conserved αVISIT‐DG sequence residue αAsp‐350 plays important functional role in the catalytic sites.MethodsMutagenic analysis of αAsp‐350 along with other catalytic site residues was performed to generate single and double mutants. Growth patterns were observed on limiting glucose and succinate plates. MgPi protection assay against NBD‐Cl induced inhibition was performed to reveal the direct or indirect role of residues in Pi binding.ResultsLately, we found that αThr‐347 of the VISIT‐DG sequence is the sixth important residue involved in phosphate binding other than βLys‐155, βArg‐182, αArg‐376, βArg‐246, and αSer‐347 while αIle‐346 and αIle‐348 are required for the maintenance of catalytic site along with βAsn‐243, αPhe‐291, and αGly‐351. Currently, we are exploring the role of negatively charged αAsp‐350 in the Pi binding sub‐domain of the catalytic sites of ATP synthase.Support or Funding InformationThis work was supported by the National Institutes of Health Grant GM085771 and A.T. Still University‐Kirksville College of Osteopathic Medicine Biomedical Science Graduate Program Funding #850‐611.
The Ribosome‐Associated Complex Suppresses [PSI+] Prion Formation in Yeast
Authors: Kelly Christina , Tessitore Thomas , Taddeo Jessica , Cameron Dale M 
Published: November 2023

Prions are self‐propagating infectious protein conformations. In mammals prions underlie the transmissible spongiform encephalopathies, a set of invariably fatal neurodegenerative disorders associated with misfolding of the mammalian prion protein, PrP. In recent years increasing numbers of fungal proteins capable of infectiously propagating altered conformations have been discovered. To gain insight into the mechanisms by which cells regulate prion formation, propagation and toxicity, we previously examined the role of the ribosome‐associated complex (RAC) chaperones in the formation and toxicity of the [PSI+] prion in yeast. The [PSI+] prion results from a self‐propagating amyloid form of the essential translation termination factor Sup35, and is manifested phenotypically as elevated levels of nonsense suppression. The RAC consists of the Hsp70 chaperone Ssz1 and the Hsp40 chaperone Zuo1, which anchors the complex onto ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. Previously we showed that yeast cells lacking Zuo1, and thus lacking RAC function on ribosomes, exhibit higher frequencies of spontaneous and induced [PSI+] formation. Cells expressing variants of Zuo1 that are unable to associate with ribosomes, or that are unable to stimulate Ssb ATPase activity, exhibit similarly high levels of prion formation. These findings are consistent with a role for the RAC in chaperoning nascent Sup35 to prevent misfolding of the N‐terminal prion domain as it emerges from the ribosome. Zuo1 can additionally function as transcriptional co‐activator for the pleiotropic drug resistance pathway, which enhances resistance to various drugs and environmental toxins. Its role in this pathway requires unfolding of the C‐terminal domain of Zuo1, which prevents its association with ribosomes. Since a variety of environmental stress conditions are known to induce prion formation, RAC dissociation from ribosomes under these conditions may facilitate prion formation. Our current work aims to understand the mechanism by which Zuo1 dissociation from ribosomes is triggered in response to environmental stress.Support or Funding InformationThis work was supported by an award from the National Institutes of Health National Institute of General Medical Sciences to DMC.
The Role of ER‐resident Lectin Chaperone UGT1 in MHC Class I Peptide Loading
Authors: Arshad Najla , Cresswell Peter 
Published: November 2023

The phenomenon of antigen processing and presentation aids the immune system in distinguishing between normal and diseased cells. Antigen presentation by MHC class I heavy chain‐β2m heterodimers (MHC‐I) to CD8+ T cells requires association of MHC‐I with peptides, a process that occurs in in the ER. Binding is facilitated by the Peptide Loading Complex (PLC), which consists of the TAP transporter, the ‘peptide editor’ tapasin, which is disulfide linked to the thiol‐oxidoreductase ERp57, and a lectin chaperone, calreticulin (CRT). Heterodimers without peptides are inherently unstable and are not expressed on the cell surface. Even when MHC‐I associates with a peptide, only those molecules loaded with a peptide deemed “optimal” by quality control mechanisms in the ER proceed to the cell surface.Proteins involved in this quality control include the ER‐resident lectin chaperones calnexin and CRT that interact with MHC‐I via its conserved N‐linked glycan. Once the peptide‐associated MHC‐I leaves the PLC, it can be a substrate for the enzyme UDP‐glucose:glycoprotein glucosyltransferase 1 (UGT1), which selectively reglucosylates the N‐linked glycan of the MHC‐I heavy chain. The glucosylated MHC‐I can then re‐associate with CRT in the PLC, allowing for additional rounds of peptide binding. This process is reminiscent of the ubiquitous calnexin‐calreticulin cycle and has been adapted by the cell for optimal antigen presentation by MHC‐I. However, in this case, MHC‐I is a folded and functional molecule.This study aims to understand the mechanism by which UGT1 recognizes a peptide/MHC‐I complex. Biochemical assays using purified UGT1 and the human MHC‐I allele HLA‐A2 unexpectedly showed that there is no strong correlation between the affinity of the associated peptide and reglucosylation of the HLA‐A2 glycan. Recognition may be based on conformational changes in MHC‐I induced by the peptide. Consistent with this, published hydrogen/deuterium exchange studies show that such changes occur near the glycosylation site, and are independent of the affinity of the peptide for MHC‐I.Support or Funding InformationCancer Research Institute Irvington Fellowship and The National Institutes of Health:R01AI09720605
Functional characterization of natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible 70kDa heat shock gene in humans
Authors: Oliverio Ryan , Nikolaidis Nikolas 
Published: November 2023

The cellular stress response system is responsible for maintaining cellular homeostasis during periods of stress caused by environmental insults including pathogens and disease. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. Therefore, we hypothesized that natural occurring variations in the sequence of Hsp70s will affect cellular and subsequently species adaptation. To investigate the relationship between adaptation, disease susceptibility, and Hsp70s' variation we tested whether natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter its function. Specifically, the wild‐type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site‐directed mutagenesis. We then determined whether any of these mutations affected the intracellular localization of HSPA1A. These experiments were performed by using GFP‐tagged HSPA1A and fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, and visualized using confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Furthermore, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly‐glutamine carrying huntingtin proteins. This assay determined that one of the mutations caused increased cell death as compared to the WT, suggesting that it may alter HSPA1A's ability to prevent protein aggregation. We also determined whether these mutations affect the ability of HSPA1A to refold heat‐denatured luciferase. These assays revealed that only one mutation resulted in significantly lower levels of refolded enzyme, suggesting a putative loss‐of‐chaperone function. Lastly, live‐dead assays revealed that human cell lines carrying two of these mutations had significantly less resistance to heat and ethanol stress than cells expressing the WT protein. Given that these natural variants are found at a very low frequency in humans we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress.Support or Funding InformationThis project was supported by funds from NIH and CSUF to NN
Dual function of the trigger factor chaperone in nascent protein folding
Authors: Kaiser Christian , Maciuba Kevin , Liu Kaixian 
Published: November 2023

Multi‐domain proteins often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. Using optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, we find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the nascent chain‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding these early folding defects is crucial, since they can propagate and result in misfolding of the entire protein.Support or Funding InformationPew Scholars Program