bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution and natural variation of HSPA1A, the major stress inducible gene, in humans
Authors: Nguyen Peter , Kdeiss Brianna , Ord Sara , Hess Kyle , Oliverio Ryan , Nikolaidis Nikolas 
Published: November 2023

HSPA1A is the major stress inducible 70‐kDa Heat shock protein (Hsp70) in humans. This protein is a critical component of the cellular stress response and alterations in its function have been associated with several disease phenotypes, such as cancer, and neurodegenerative conditions. However, the extent of HSPA1A's natural variation within and between species remain largely unknown. Therefore, we investigated how this gene evolved in humans and other mammals. Our results reveal that in all Eutherian mammals HSPA1A is actually two almost identical proteins coded by two tandemly arranged genes. This extreme level of sequence conservation, both at the nucleotide and protein levels, can be explained by a gene conversion‐like mechanism that resulted in sequence homogenization. However, gene conversion alone cannot explain the observed evolutionary pattern of all the mammalian heat‐inducible genes, which contain several non‐amino acid altering (synonymous) mutations. Therefore, we conceptualized that both gene conversion and purifying selection act to conserve these Hsp70 genes. This notion is also supported by analyses of HSPA1A variation in humans, which revealed that all mutations have a very low frequency and that the majority of these mutations is synonymous. The presence of purifying selection was further strongly implied by determining whether natural variants affect the major chaperone functions of HSPA1A. Specifically, recombinant proteins corresponding to the wild‐type (WT) and five mutated variants were tested for their ability to hydrolyze ATP. These experiments revealed that the mutations did not significantly alter the rate of ATP hydrolysis. Then Isothermal Titration Calorimetry was employed to test whether the mutations alter binding of HSPA1A to ATP, ADP, and protein substrates. These experiments showed that only one mutation increased the entropy of the reactions. Lastly, the effects of these mutations on protein stability was determined by calculating the protein's melting temperature (Tm). This assay revealed that three mutations had significantly higher Tm as compared to the WT protein. These experiments suggest that although purifying selection acts to preserve the primary functions of HspA1A, other secondary functions of the molecule may be altered. Collectively, our results constitute an evolutionary paradigm in which gene conversion, purifying selection, and neofunctionalization act conversely to conserve a critical component of the heat shock response.Support or Funding InformationThis project was supported by funds from NIH and CSUF to NN
Identification of a pharmaceutical therapeutic for Nod2, a protein mutated in Crohn's Disease, through development of a screen using split GFP complementation
Authors: Wastyk Hannah Constance , Hou Ching Wen , Grimes Catherine 
Published: November 2023

The human body houses over 10 trillion bacterial cells to make up our microbiome. These bacteria can be commensal or pathogenic, and it is the role of our innate immune system to delineate between the two. Innate immune receptors, such as Nod2, are responsible for binding to bacterial cell wall fragments. Nod2 binds to bacterial cell wall fragment, muramyl dipeptide (MDP), which leads to subsequent downstream activation of the NF‐κB pathway and a protective inflammatory response. Mutations in Nod2 are correlated with an increased susceptibility to Crohn's disease. Nod2 mutations that decrease its stability disrupt the downstream signaling response. Initial work identifies a chaperone protein, Hsp70, which binds to Nod2 and assists its stability and activity. The interaction of Nod2 and Hsp70 was characterized to suggest that Hsp70's ATPase activity is dispensable for Nod2 stabilization. A truncated 71 amino acid domain of Hsp70 was shown to bind and stabilize Nod2 (wild type and Crohn's variant). Here, we go a step further by expressing the minimized peptide chain in cells and measuring the relative signaling response via a NF‐kB luciferase assay. The 71 minimized peptide chain was also used as a pharmacological tool through the principle of split Green Fluorescent Protein (GFP) complementation for development of a screen for pharmaceutical therapeutics. The peptide and Nod2 were tagged and detected with engineered self‐assembling fragments of GFP. When the peptide and Nod2 are bound, fluorescence is detected. Each pharmacoperone was individually incubated with the fluorescent protein complex. Effective pharmacoperones should competitively bind to Nod2, and thus disrupt the binding of the peptide and lead to lower fluorescence. The most promising pharmacoperones from the screen will be further analyzed for Nod2 stability and signaling in cell based assays. Successful identification of a pharmacoperone that mimics the natural stabilization of Hsp70 can be applied to restore function of a number of disease‐relevant mutated proteins.Support or Funding InformationThe authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.
Loss of Sil1, an ER Co‐Chaperone, Causes an Age‐Dependent Collapse of Skeletal Muscle Proteostasis, Affecting Pathways Critical for Muscle Physiology
Authors: Ichhaporia Viraj P. , Vogel Peter , Frase Sharon , Horner Linda , Hendershot Linda M. 
Published: November 2023

ObjectiveDecipher molecular mechanism(s) underlying the myopathy associated with Marinesco‐Sjögren syndrome.BackgroundMarinesco‐Sjögren syndrome (MSS) is an autosomal recessive, multisystem disorder clinically characterized by cerebellar ataxia, bilateral cataracts, and progressive myopathy. MSS is attributed to loss‐of‐function mutations in Sil1, a nucleotide exchange factor for the ER resident Hsp70 molecular chaperone‐BiP.MethodsUsing a combination of an inverted screen test, histopathology, electron microscopy, LC‐MS/MS‐based proteomics, real‐time PCR, and western blotting, we investigated the effects of Sil1 disruption in skeletal muscles of the MSS mouse model (Sil1Gt).ResultsSil1Gt mice exhibited progressive loss of skeletal muscle mass and strength beginning around six months of age. Histopathological and ultrastructural investigations confirmed a multifocal, myogenic myopathy which predominantly affected fast myofibers and phenocopied the MSS‐myopathy. Electron microscopy (EM) revealed a severe swelling of sarcoplasmic reticular triads, autophagic vesicles, electron‐dense material suggestive of protein aggregates, and disruption of the overall cytoarchitecture.Quantitative proteomic profiling performed on quadriceps from six‐month‐old Sil1Gt and wild‐type mice identified that 490 proteins were significantly altered, including secretory pathway proteins, as well as proteins residing in all major cellular organelles. Importantly, multiple pathways critical to muscle physiology were strikingly affected.To validate these changes in protein expression and to determine their onset and progression, we performed western blotting and mRNA expression analyses on quadriceps derived from mice ranging from 1–18 months of age. Prior to the onset of muscle weakness, we detected increased levels of BiP and the lectin chaperones, accompanied by activation of the unfolded protein response, which became profound after six months. We observed robust increases in markers of autophagy, lysosomal biogenesis, and cytoskeletal remodeling. Conversely, components of glucose metabolism and pentose phosphate pathways were dramatically decreased. Interestingly, while levels of Insulin and Igf1 receptors ‐which are synthesized in the ER‐ were decreased, there was a progressive increase in the insulin‐regulated glucose transporter, Glut4. These changes were accompanied by increased levels of both total and phospho‐Akt, similar to data observed in murine skeletal muscles with genetic ablation of both the Insulin and Igf1 receptors.ConclusionThese findings provide evidence for extensive cellular perturbations upon loss of Sil1 in murine skeletal muscles due to a wide‐spread proteostasis collapse, offering critical insights into pathomechanisms underlying the MSS‐myopathy.
Chaperoning the Proteome
Authors: Houry Walid A 
Published: November 2023

Molecular chaperones are critical to maintaining cellular protein homeostasis. There are several families of chaperones in the cell with some being specific for certain substrates, while others act on many protein targets. As a result, chaperones are expected to be involved in many cellular pathways with only a few being well elucidated. To obtain a comprehensive view of chaperone function in the cell, we developed an integrative approach based on systematic physical and genetic interaction mapping to decipher interactions involving all main chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. The analysis revealed novel chaperone substrates and novel chaperone activities. As part of this analysis, we unexpectedly found that many interactors of the Hsp90 chaperone system are proteins that form foci under stress conditions. Such foci forming tendency was then demonstrated for the highly conserved AAA+ ATPases Rvb1 and Rvb2 that are part of the R2TP complex that interacts with Hsp90. In response to nutrient deprivation, a fraction of Rvb1 and Rvb2 colocalize to perinuclear foci that dissolve upon nutrient addition. We propose that foci formation might be a feature of many chaperone substrates.Support or Funding InformationCanadian Institutes of Health Research
Thiol Addition to Conjugated Nitrolinoleic Acid
Authors: Alvarez Beatriz , Turell Lucia , Vitturi Dario A , Coitiño Elena Laura , Lebrato Lourdes , Moller Matias N , Sagasti Camila , Salvatore Sonia R , Woodcock Steven R , Schopfer Francisco J 
Published: November 2023

Nitroalkene fatty acids are formed endogenously after nitration of unsaturated fatty acids and exert protective effects via reversible Michael addition to thiol‐containing proteins in signaling pathways. Conjugated linoleic acid is nitrated preferentially and forms 9‐ and 12‐nitro‐octadecadienoic acid (NO2‐CLA). In this work, we characterized the reactivity of NO2‐CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine and β‐mercaptoethanol) and with human serum albumin (HSA). Reactions with low molecular weight thiols followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2‐CLA. LC‐UV‐Vis and LC‐MS analyses showed that the fast reaction corresponds to the formation of the β‐adduct (the kinetic product) while the slow reaction corresponds to the δ‐adduct (the thermodynamic product). The rates (kon and koff) and equilibrium constants (Kd) for both reactions were determined. The pH dependencies of the rate constants, the correlations between intrinsic reactivity and thiol pKa and the absence of deuterium solvent kinetic isotope effects suggest stepwise mechanisms with thiolate attack on NO2‐CLA to form the anionic intermediates as rate‐controlling steps. The characteristics of NO2‐CLA, of the transition states and of the anionic intermediates were further investigated through computational modeling. In human urine, cysteine‐δ‐adducts were detected, which is consistent with the higher stability of the δ‐adducts. HSA was found to bind NO2‐CLA non‐covalently and to slowly form covalent adducts at Cys34. Our results provide new insights into the mechanisms that mediate not only the signaling actions of NO2‐CLA, but also its transport and storage.Support or Funding InformationThis work was supported by grants and fellowships from CSIC (Universidad de la República, Uruguay) (to LT, MNM and BA), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) (to LL), NIH grants K01‐HL133331 (DAV), R01‐AT006822 (FJS) and AHA #14GRNT20170024 (FJS).
Differential Protein Splicing of Salt Dependent Inteins from Haloquadratum Walsybi
Authors: Lynch Abigeal Kathleen , Amunya Shirley , Reitter Julie , Mills Kenneth 
Published: November 2023

Protein splicing is a self‐catalyzed, post‐translational process in which an intervening polypeptide (intein) catalyzes its own removal from the flanking polypeptides (exteins), as well as the ligation of the exteins. We are interested in the splicing of two inteins interrupting the cdc21 protein from Haloquadratum walsbyi (Hwa). Hwa is a halophilic archaeon. Because of this, we hypothesized that protein splicing would be salt dependent. The first intein, Cdc21a, does not splice on overexpression in E. coli, but can be induced to splice in vitro on incubation with 2.5 M sodium chloride at 28°C. The second intein, Cdc21d, splices in vivo, and further splicing can be induced in vitro with higher concentrations of salt. Although the first intein requires 2.5 M salt to splice, Cdc21d requires only 500 mM salt to induce additional splicing. Given that both inteins interrupt the same protein, this differential activity could result in different protein products due to alternative splicing in the full precursor. We are studying this relative activity when the two inteins are expressed in the same precursor fusion protein.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
A structural investigation and splicing study of inteins from Halobacteria
Authors: Zhang Wanlin , Lynch Abigeal K. , Gomez Alvin O. , Reitter Julie N. , Mills Kenneth V. 
Published: November 2023

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the flanking protein while simultaneously ligating the two flanking exteins. Inteins can be found in many types of archaea, such as halophiles, which specifically thrive in environments with high salt concentrations. The lab previously reported that the Halobacterium salinarum (Hsa) DNA polymerase II (Pol II) intein promotes conditional protein splicing at high concentrations of sodium chloride. To study the structure of the intein, we attempted to find conditions for the formation of protein crystals of the unspliced Hsa fusion protein under conditions of different precipitating solutions. Furthermore, we also attempted to learn if splicing of an intein interrupting the Replication Factor C (RFC) from Haloquadratum walsbyi (Hwa) is also salt dependent.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
Purification and Characterization of Flavin Reductase, DszD, from Rhodococcus erythropolis.
Authors: Mendez Gilberto 
Published: November 2023

Flavin reductases are found in all organisms, and catalyze essential reactions in all domains of life. Flavins such as flavin mononucleotide, flavin adenine dinucleotide, and riboflavin are essential for many metabolic processes and are best known for their role in oxidation‐reduction reactions. We are working on crystallizing and assessing the catalytic capabilities of the NADH‐dependent flavin reductase, DszD, from the biodesulfurizing bacterium Rhodococcus erythropolis. In our work, DszD has been successfully overexpressed and purified essentially to homogeneity from E. coli. We have verified the substrate preferences of DszD, showing the enzyme has maximal activity with flavin mononucleotide. DszD was subjected to a sparse matrix crystallization experiment using the hanging drop vapor diffusion method. Promising crystallization conditions were identified, replicated, and are currently being optimized to improve crystal quality. Once solved, the structure of DszD will lead to the identification of the active site residues important for substrate binding and catalysis.Support or Funding InformationThis work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500.
Using Dynamics and Structure to Understand Allostery in Signaling Enzymes
Authors: Peti Wolfgang 
Published: November 2023

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in phosphorylation enzymes, specifically in the protein tyrosine phosphatase PTP1B and in serine/threonine kinases. Only by combining a large array of biophysical tools with NMR having a leading role, it is possible to unravel how these enzymes are regulated and function. We will present how dynamics at different timescales drive the enzymatic cycle of these enzymes as well as show how allostery controls their enzymatic activity.Support or Funding InformationThis work was supported by the American Diabetes Association Pathway to Stop Diabetes Grant 1‐14‐ACN‐31, NIH grants R01GM098482, R01NS091336 and grants from the Lundbeck and Novo Nordisk Foundations.
An Essential Oil Blend Specifically Enhances Immune Responses in Human Cancerous Co‐Cultures
Authors: Han Xuesheng , Parker Tory 
Published: November 2023

Essential oils, composed of aromatic compounds naturally found in plants, have been used in folk medicines to prevent and treat a variety of diseases in humans all over the world for many hundreds of years. Despite growing scientific evidence that essential oils possess important therapeutic benefits, research on their biological activities in complex human disease models is scarce. To contribute to this understanding, we analyzed the biological activities of an essential oil blend (EOB) in validated human cell co‐cultures, with or without tumor cells. These disease models allow for measurement of changes in protein biomarkers induced by EOB treatment. The EOB showed significant effects on important biomarkers related to inflammation, immune system function, wound healing, and tumor biology. In tumor co‐cultures, the EOB elevated inflammatory and immune‐related biomarkers, including sIL‐17A, sIL‐2, sIL‐6, VCAM‐1, CD‐40, CD‐69, sGranB, and sIFNγ, but many of these same biomarkers were decreased by the EOB in non‐tumor cell co‐cultures, suggesting that the EOB shows tumor‐specific immune enhancement. The EOB also broadly impacted genome‐wide gene expression in a dermal fibroblast cell model, as measured via total mRNA microarray assays. Finally, the EOB also pleiotropically regulated multiple signaling pathways in human cells, such as the hepatic fibrosis/hepatic stellate cell activation pathway, the antigen presentation pathway, the mitotic roles of polo‐like kinase pathway, and the cyclins and cell cycle regulation pathway. In summary, the study provided evidence suggesting that the EOB can beneficially impact human cells through anti‐inflammatory activities, immune enhancing functions, modulation of wound healing, and anticancer properties.Support or Funding InformationThis study was funded by dōTERRA International LLC, Pleasant Grove, UT, USA.
Mechanism and in vivo Activity of a Covalent Inhibitor of ERK Docking
Authors: Dalby Kevin N , Anslyn Eric V , Zamora‐Olivares Diana , Kaoud Tamer 
Published: November 2023

ERK is a member of the mitogen‐activated protein kinase (MAPK) family, which consists of three main classes of enzyme; the extracellular signal regulated kinases (ERKs), the Jun N terminal kinases (JNKs), and the p38 MAPKs. Melanoma is often driven by ERK pathway activation, with high ERK activity, a hallmark of melanomas harboring activating BRAF and NRAS mutations. Vemurafenib and dabrafenib are BRAF inhibitors, and trametinib is a MEK inhibitor used for the treatment of BRAF mutant melanoma. These drugs are clinically effective in the short term and result in ERK pathway inhibition. Importantly, a high degree of ERK inhibition correlates with clinical response (tumor shrinkage). ERK pathway reactivation is a hallmark of many melanomas that have acquired resistance to BRAF and MEK inhibitors and consequently progress. Therefore, it is of interest to develop novel methods to inhibit ERK activity directly. ERK signaling is mediated by a critical protein docking site that lies outside the catalytic domain. We describe a small molecule inhibitor of ERK that binds this docking site covalently, to suppress ERK signaling in drug resistant melanoma cells and in a xenograft model of melanoma. We provide detailed characterization of its mechanism of inhibition and also describe new approaches for the quantification of ERK activity in tumor samples using fluorescent sensor arrays.Support or Funding InformationRP 140648, RP 140649, RP160657, Cancer Prevention Research Institute of Texas (CPRIT)
Characterization of a cardiac drug‐inactivating enzyme from the prominent human gut microbe, Eggerthella lenta
Authors: Koppel Nitzan , Bisanz Jordan , Turnbaugh Peter , Balskus Emily P 
Published: November 2023

Metabolism of pharmaceuticals by the human gut microbiota has been appreciated for several decades. Currently, over 50 examples are known in which the pharmacological properties, bioactivities, and toxicities of a drug are altered by the gut microbiota. However, only a few of these examples have been linked to specific bacteria or enzymes, and the precise mechanisms of these transformations remain largely unknown. The gut microbiota harbors approximately 150× more genes than the human genome providing additional metabolic capacity to modify drugs beyond host metabolism. In particular, gut microbial chemistry tends to be reductive and hydrolytic, in contrast to oxidative and conjugative host metabolism of xenobiotics. Additionally, the composition of the microbiota can vary substantially among individuals, leading to differences in drug metabolism. A deeper knowledge of microbial drug metabolism at the molecular level is thus crucial for understanding variability in patient response to drugs, and improving efficacy in the clinic.In our work, we biochemically confirmed that Cgr2, a putative cardiac glycoside reductase from the prominent gut microbe Eggerthella lenta, is responsible for reduction and inactivation of the widely used cardiac drug, digoxin. Cgr2 is a divergent homolog of FAD‐dependent fumarate reductases and harbors Fe‐S clusters with non‐canonical binding motifs that could not be predicted computationally. Bioinformatics and network analyses also revealed that Cgr2 is a highly unique enzyme that likely employs a novel mechanism of substrate binding and reduction. Through microbial isolation and sequencing of human‐associated isolates, we determined that the cgr2 gene is found exclusively in a subset of E. lenta strains, and is remarkably conserved at the amino acid sequence level (>95% identity). Digoxin metabolism does not enhance E. lenta growth, and more work is required to understand the physiological implications of this highly conserved gene. We are also actively working to determine the mechanism of substrate reduction through biochemical and structural methods.Support or Funding InformationFunded by Harvard University, National Science Foundation, Packard Fellowship for Science and Engineering, George W. Merck Fund.