bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alteration of miR‐186 expression modifies inflammatory markers in normal epithelial and prostate cancer cell models
Authors: Suman Suman , Jones‐Reed Dominique Z. Z. , Schmidt M. L. , Clark Geoffrey J. , Klinge Carolyn , Barve Shirish , Kimbro Kevin S. , Kidd La Creis R. 
Published: November 2023

Dysregulation of miRNAs and chronic inflammation are strongly implicated in the development of various malignancies, including prostate cancer (PCa). Previously, our lab identified several inflammatory and immune response sequence variants in CCL5 and CCR5 significantly modified PCa risk. Recently, we propose an oncogenic role for miR‐186‐5p based on its up‐regulation in serum from PCa patients and metastatic PCa cell lines. Moreover, miR‐186‐5p inhibition reduced proliferation, anchorage independent growth and invasion in metastatic PC cells (PC3, MDA‐PCA‐2b). We hypothesize dysregulation of inflammatory and immune response markers may enhance immune surveillance leading to a reduced aggressive tumor phenotype. Following modulations in miRNA‐186 levels in normal prostate epithelial (RWPE1) and metastatic PCa (PC3) cell lines, we evaluated alterations in mRNA expression using micro‐array analysis. Ectopic expression of miRNA‐186 in the RWPE1 resulted in a 1.3–2.6‐fold down‐regulation in TLR2, IRAK2, CCL20, IL1RAP, IL1RAP, IL15, and IL17RD (FDR p‐value ≥7.8×10−06). Whereas, inhibition of miR‐186 in PC3 cells corresponded with a 1.4‐fold up‐regulation of IL18R1 (p=0.0095). Notably, these inflammatory markers are involved in cell invasion (TLR2), apoptosis (IL17RD), tumorigenesis (TLR2, IRAK2) and autoimmune diseases (CCL20). Validation of these targets is underway using qRT‐PCR, western blots and luciferase reporter assays. We will assess the mechanism by which these miR‐186 targets suppress aggressive cancer behavior using knock‐out and knock‐in cell and ultimately animal models. Future studies may identify inflammatory targets that may guide immune therapies for the effective treatment of aggressive prostate cancer.Support or Funding InformationJames G. Brown Cancer Center, Our Highest Potential endowment in cancer research
Investigation of PAX3‐FOXO1 Effect on NEAT1 Expression in Alveolar Rhabdomyosarcoma Cells
Authors: George Victoria Agbeke , McDaniel Brianyell , Johanson Kelly E. 
Published: November 2023

Rhabdomyosarcoma (RMS) is pediatric sarcoma in the soft muscular tissue. One of the two major types of rhabdomyosarcoma is alveolar rhabdomyosarcoma (ARMS). ARMS generally occurs in muscles in the abdomen, arms, and legs. Due to its tendency to grow more rapidly, it is a more aggressive form of the cancer that has a higher risk factor than other forms of RMS. The expression of PAX3‐FOXO1 in ARMS may contribute to the formation of tumors by inducing tumor growth activities or inhibiting tumor suppressing activities. There are many studies investigating targets of PAX3‐FOXO1 and some indicate that PAX3‐FOXO1 binds to long noncoding RNA (lncRNA), although it is not known if this interaction affects the expression of the lncRNA. Nuclear‐enriched autosomal transcript 1 (NEAT1) is a 4kb lncRNA that potentially is bound by PAX3‐FOXO1. Several putative PAX3‐FOXO1 binding sites are present in the DNA fragment of NEAT1 that was identified in a ChIP assay. We are examining the NEAT1 sequence to narrow down the specific site of PAX3‐FOXO1 interaction as well as determining the effect of PAX3‐FOXO1 on NEAT1 expression. The expression of NEAT1 is necessary for differentiation of muscle cells and a change in this expression due to PAX3‐FOXO1 may be linked to ARMS development. Finding the effect of PAX3‐FOXO1 on expression of NEAT1 will help to uncover more information about the mechanisms involved in the development and progression of ARMS.Support or Funding InformationNIH BUILD TL4GM118968, NIH BUILD RL5GM118966‐02
The Role of a Flexible loop in Metal transfer between periplasmic zinc proteins
Authors: Fullam Stephanie Hope 
Published: November 2023

Zinc is an essential nutrient, and proteins mediating zinc acquisition are important virulence factors among pathogenic bacteria, making them attractive targets for the development of novel antibiotics. Of particular interest are the ATP binding cassette (ABC) transporters, which utilize extracellular solute binding proteins (SBPs) to bind zinc with high affinity and specificity for delivery into the cell via integral membrane permeases. We are studying the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria. Using a simple fluorescence assay, we have found that the zinc SBP AztC can acquire zinc from solution or from a metallochaperone, AztD. The crystal structure of AztC shows that, like all known zinc‐specific SBPs, this protein has a flexible loop near the zinc binding site. We hypothesized that this flexible loop is required for the recognition and subsequent transfer of zinc from its chaperone AztD. To test this, we generated a deletion mutant lacking the flexible loop (Δloop AztC) and tested zinc acquisition from solution and from AztD. Consistent with our hypothesis, the affinity of the mutant protein for zinc in solution is unaffected, yet it can no longer acquire zinc from AztD. In order to refine our understanding of those residues required for AztD recognition and zinc transfer, several site directed mutants of conserved loop residues were generated and evaluated for their ability to acquire zinc from AztD. Three His residues appeared to be absolutely required for zinc transfer, while mutation of a conserved Asp had no effect. Tyr121 appeared to be dispensable for zinc transfer, although the process was significantly slower for the Y121A mutant relative to WT. A detailed understanding of the mechanisms governing extracellular zinc management in bacteria may eventually lead to the development of novel antibiotics targeting this important process.Support or Funding InformationNIH (National Institutes of Health) grant number: 1sc2gm111170‐01
Molecular mechanisms associated with early onset primary dystonia (DYT16) caused by mutations in PACT
Authors: Burnett Samuel B , Vaughn Lauren , Patel Rekha 
Published: November 2023

Dystonia is an inherited neuromuscular movement disorder that has been shown to result from mutations in 25 different genes and classified into 21 categories. The resultant phenotype consists of both agonist and antagonist muscles firing simultaneously. Consequently, patients suffer from inability to control their dystonic limb, often sustained repetitive movements, and have a compromised posture. One type of primary dystonia, the early onset dystonia 16 (DYT16), has been shown to be result from various mutations in the PACT (also known as PRKRA) gene that has been well characterized for its role in regulating apoptosis in response to cellular stress.Under conditions of viral, oxidative, or endoplasmic reticulum stress, the cellular apoptosis has been shown to be regulated by prolonged translation inhibition resulting from kinase activity targeting the eukaryotic translation initiation factor 2 alpha (eIF2a). Events leading to eIF2a phosphorylation in response to cell stress are attributed to interactions between three double stranded RNA binding proteins: PACT, TRBP, and PKR. These three proteins interact with each other forming homo‐ and heterodimers to impact cellular fate in response to stress signals. PACT is a protein activator of PKR, a serine‐threonine kinase involved in innate immunity. PACT‐PKR interaction is essential for facilitating apoptosis; whereas, transactivation RNA binding protein (TRBP) serves as a PKR inhibitor via PACT‐TRBP and TRBP‐PKR heterodimerization. These three proteins share evolutionarily conserved double stranded RNA binding motifs that also facilitate protein‐protein interactions and regulate the cell survival and apoptosis.We investigated the altered protein‐protein interactions between PACT, PKR, and TRBP that result from various mutations reported in DYT16 patients, and for the first time in a mouse model of DYT16. Using various biochemical techniques, we characterized how these mutations influence PACT's function during stress response to regulate apoptosis. By utilizing DYT16 patient samples as well as an analogous mouse model we are attempting to better understand how PACT mutations may lead to early onset dystonia. Our research highlights the insight obtained by applying biochemical, and molecular analysis to patient cells and mouse models to understand the etiology and pathophysiology of dystonia.Support or Funding InformationThe Dystonia Medical Research Foundation
Bacteriophytochromes in myxobacteria: Implications for light‐control of cell development
Authors: Tracy Gregory Cornell , Gallagher Kevin D , Varela Joseph D , Bizhga Dorina , Duong Phu , Nugent Angela , Stojkovic Emina A 
Published: November 2023

Bacteriophytochromes (BphPs) are red‐light photoreceptor proteins found in photosynthetic and non‐photosynthetic bacteria. They are unique among photoreceptors in their ability to undergo reversible photoconversion between spectrally distinct red (Pr, 700nm) and far‐red (Pfr, 750nm) light absorbing states. BphPs are composed of an N‐terminal photosensory module, consisting of PAS, GAF, and PHY domains, covalently linked to an effector domain, usually a histidine kinase. For photoresponse, BphPs require biliverdin (BV), an open‐chain tetrapyrrole covalently attached to an N‐terminal conserved cysteine. BphPs of the non‐photosynthetic myxobacterium Stigmatella aurantiaca are of special interest. Myxobacteria are distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies, which in Stigmatella aurantiaca is controlled by light. The two BphPs from S. aurantiaca, SaBphP1 and SaBphP2, have distinct photochemistry, although they bind the same bilin chromophore and share a large sequence identity. Unlike classical BphPs, wild‐type SaBphP1 lacks a conserved histidine (His289) that stabilizes the bilin chromophore and undergoes limited Pr/Pfr photoconversion, that can be restored by a single Thr289His mutation in the bilin‐binding domain. The crystal structures of SaBphP1 wild‐type and Thr289His mutant differ in the conformation of the chromophore binding cavity. Furthermore Cystobacter fuscus, from the same suborder of myxobacteria as S. aurantiaca, contains three BphPs, two of which are lacking the conserved His. CfBphP1, like SaBphP1, contains a Thr in place of the conserved His and shows the same photochemistry. In CfBphP3 the conserved His is replaced by a glycine (Gly) which results in a complete lack of Pr/Pfr photoconversion. Therefore, we hypothesized that if we mutate Thr in SaBphP1 and His in SaBphP2 to Gly we can suppress photoconversion of these proteins. Surprisingly, we observed complete recovery of Pr/Pfr photoconversion in SaBphP1 Thr289Gly although reversion to the thermally stable, dark‐adapted Pr state was shorter than the His mutant. Currently, we are in the process of characterizing SaBphP2 mutant where the conserved His is substituted by a Gly. Our structural and sequence analyses of S. aurantiaca's and C. fuscus BphPs highlight critical His289 interactions with the chromophore and suggest the function for BphPs in fruiting myxobacteria.Support or Funding InformationNSF MCB RUI 1413360
Lactoferrin interacts with SPLUNC1 to amelioate lipopolysaccharide‐induced inflammation of human nasal epithelial cells
Authors: Chen Chuan‐Mu , Tsou Yung‐An , Chen Hsiao‐Ling 
Published: November 2023

Chronic rhinosinusitis is a chronic inflammatory disease caused by a chronic bacterial infection that last over 3 months. Innate immune deficiency is considered a crucial factor in the etiology of mucosa disease. The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an important innate material in the upper airway, and lactoferrin (LF) aids the innate functions in humans. In this study, an nasal epithelial model was used to investigate how LF modulates SPLUNC1 to reduce the inflammatory process mediated by lipopolysaccharide (LPS). The inflammation of human RPMI‐2650 cells was induced by LPS to evaluate SPLUNC1 expression after treating the cells with bovine LF (bLF). The interaction pathway between LF and SPLUNC1 in LPS‐induced inflammation was further investigated. Our study reveals that the addition of bLF results in the recovery of SPLUNC1 expression in nasal epithelial cells under LPS‐induced inflammation. MAPK is involved in the main pathway for the SPLUNC1 and bLF interaction. Decreased SPLUNC1 function could be recovered by addition of bLF. The MEK1/2‐MAPK signaling pathway is crucial for the SPLUNC1 and bLF interaction. Therefore, LF could support SPLUNC1 in the innate immunity recovery process.Support or Funding InformationThis research was supported in part by grants MOST‐104‐2313‐B‐005‐043‐MY3 and MOST‐105‐2314‐B‐039‐030 from the Ministry of Science and Technology, Taiwan.
Thermodynamic partitioning forces at the membrane protein interface
Authors: Mahalakshmi Radhakrishnan 
Published: November 2023

Transmembrane proteins fold by the orchestrated interplay of membrane chaperones, holdases and the lipid membrane. Upon folding, membrane protein systems attain a thermodynamically stabilized framework through optimized energetics involving protein – membrane – solvent interactions. In particular, the water‐bilayer interface demands selectively positioned amino acids to arbitrate interactions between the hydrophobic interior and polar phospholipid headgroups. A fundamental question that arises is the extent to which the amino acid side chain contributes to membrane protein folding and in maintaining protein‐lipid interactions at the intricate interface environment of membrane proteins. Our goal is to experimentally deduce the partitioning cost of amino acids at the interface, and the associated physical principles governing the overall scaffold stability.Interface residues are important contributors for membrane protein folding, and additionally serve as post‐folding membrane anchors. In this study, we have quantified the free energy change associated with the chemical nature of the residue at the membrane interface. We achieve this by studying the unassisted folding equilibrium of the 8‐stranded transmembrane β‐barrel enzyme PagP in phosphocholine lipidic micelles and lipid vesicles. We present the first experimentally measured whole‐protein free energy scale for side chain partitioning at the membrane interface, for all naturally abundant amino acids. We obtain differential contributions of the protein‐ and lipid‐facing interface residues to the barrel folding pathway and stability of the folded scaffold. The most favorable transfer at the amphiphilic lipid‐facing interface is for hydrophobic amino acids, whereas we observe the highest energetic cost of transfer for charged and polar groups. On the contrary, we find that small and polar residues are most favorably transferred to the protein‐facing interface, whereas hydrophobic and aromatic residues promote alternate folding pathways in PagP.Our studies establish that the side chain non‐polar accessible surface area shows a direct correlation with the partitioning free energy change at the lipid‐facing interface residue. We also find that the non‐polar accessible surface correlates inversely with the thermodynamic partitioning free energy change when the interface residue is protein‐facing. We demonstrate how PagP maintains a balance between concerted folding and hydrophobic collapse by evolutionary choice of small polar residues at the protein‐facing interface. We also identify the importance of interface polar residues to the folding pathway of β‐barrel membrane proteins.Support or Funding InformationR.M. is a recipient of the Wellcome Trust/DBT India Alliance Intermediate Fellowship. This work was supported by the Wellcome Trust/DBT India Alliance award number IA/I/14/1/501305 to R.M.
Cooperative binding of cinnamon polyphenols as activators of Sirtuin‐1 protein in the insulin signaling pathway
Authors: Brennemen Martin , Mahfouz Tarek , Stockert Amy 
Published: November 2023

Cinnamon has been shown to decrease blood glucose levels in type‐II diabetics. Analysis of cinnamon extract have determined that it contains a number of polyphenolic compounds and the antidiabetic effect of cinnamon is hypothesized to be due to those phenolic compounds. The protein target of those phenolic compounds has not been identified yet. However, Sirtuin‐1 (Sirt‐1), a deacetylase in the insulin signaling pathway, seems a likely target. To investigate this possibility, we docked several polyphenolic compounds that are commonly found in cinnamon extract to Sirt‐1 in order to determine their binding affinities and compared these to Sirt‐1 affinity for resveratrol which is not in cinnamon extract, but it is a known activator of Sirt‐1. We used Sirt‐1 structure 5BTR for the docking which contained three molecules of resveratrol with a 7‐amino‐4‐methylcoumarin (AMC)‐containing peptide in the binding site. Our results indicate a cooperative binding of resveratrol molecules when docked sequentially in the binding site with the AMC peptide. We further hypothesized that the polyphenolic compounds within cinnamon would have a similar effect. To investigate this, a second set of docking simulations was performed on the polyphenolic compounds within cinnamon extract to investigate potentially similar interactions as shown by resveratrol. These docking simulations were done using AutoDock Vina in two different trials; one using sets of three of the same molecule, and the second using sets of combinations of three molecules. Results from those trials showed similar and sometimes stronger interactions than resveratrol between Sirt‐1 and the molecules docked. Molecules with affinities at least as strong as resveratrol's were used for sequential docking runs. These results strongly suggest that Sirt‐1 is a possible target for cinnamon extract.Support or Funding InformationDepartment of Pharmaceutical and Biomedical Sciences, Ohio Northern University
Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints
Authors: Chan Yvonne H. , Venev Sergey V. , Zeldovich Konstantin B. , Matthews C. Robert 
Published: November 2023

Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole‐3‐glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long‐range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologs implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence‐structure‐fitness relationships in other common motifs.Support or Funding InformationThis research was supported by NIH GM23303 to CRM.
Characterizing the Interactions Between Mg2+ and a Periplasmic Lipoprotein Involved in Mg2+ Homeostasis in Salmonella enterica
Authors: Davie Timothy J , May John F 
Published: November 2023

The ability to acquire Mg2+ is needed for certain bacteria to cause disease due to the nutrient's importance in physiological processes. Pathogenic bacteria have proteins which help them to adapt to environments where Mg2+ is in low supply. We have identified a periplasmic lipoprotein found in Salmonella enterica, a bacteria that is a major contributor to food‐borne illness, that allows the pathogen to survive in low Mg2+ environments. The Salmonella lipoprotein of 19.7 kDa monomeric molecular weight consists of a signal peptide and a single domain of unknown function that is conserved among enterobacteria. The purpose of this research project is to determine if the protein binds Mg2+ and identify the amino acid residues that are required for the protein to function properly. We have developed a protocol to overproduce and purify the recombinant lipoprotein. Protein variants are created using site directed mutagenesis to replace residues thought to be critical for binding Mg2+. To test for whether a particular residue is required for the ability of this lipoprotein to promote growth in a low Mg2+ environment, growth of Salmonella expressing these protein variants in low Mg2+ is being assayed. This physiological assay will indicate which residues are required for the physiological function of this protein in Mg2+ homeostasis. A fluorescence assay that utilizes Mg2+ sensor 8‐hydroxyquinoline‐5‐sulfonic acid (HQS) has provided evidence that the lipoprotein is sequestering Mg2+ from solution. We observed a change in HQS fluorescence when the protein is present, indicating that HQS and the protein compete for Mg2+. The results from the HQS biochemical assay, combined with our site directed mutagenesis experiments, will provide a model for how the protein functions. The results of the project may allow for a further understanding of how Salmonella and other bacteria adapt to their environment and maintain virulence in harsh conditions.Support or Funding InformationThis research is supported by an UW‐La Crosse Undergraduate Research and Creativity Grant (TD) and a UW‐La Crosse Faculty Research Grant (JM).
Translation of heat shock proteins is regulated by poly(A)‐binding protein assembly
Authors: Katanski Christopher D , Riback Joshua , Pilipenko Evgeny , Drummond David Allan 
Published: November 2023

How do cells sense and respond to changes in the environment? During heat stress, numerous eukaryotic RNA and protein species assemble into macromolecular bodies ‐ stress granules ‐ the function of which is largely unknown. Simultaneously, cells increase transcription of genes encoding heat shock proteins, many of which are molecular chaperones. In addition to increased transcription, the mRNA for these genes is also translated more efficiently. Thus, heat shock protein production is regulated by both transcription and translation. These heat‐induced mRNAs have exceptionally A‐rich 5′ untranslated regions (UTRs), which have long been known to mediate cap‐independent translation. We observe that poly(A)‐binding protein (PABP, Pab1 in S. cerevisiae) is bound to these A‐rich UTRs during exponential growth. Pab1 is able to repress translation of its own mRNA by binding a similar A‐rich UTR. We find that Pab1 represses translation of mRNA by binding A‐rich UTRs of heat shock proteins. Further, we propose that during heat‐shock, as Pab1 assembles into heat‐induced RNA‐protein granules, Pab1 releases bound 5′ UTRs, and relieves repression. We have recapitulated Pab1‐mediated repression and heat‐induced relief with an in vitro translation system. Moreover, yeast expressing assembly‐impaired Pab1 are not able to grow at elevated temperature; this result is consistent with disruption of an assembly‐mediated regulatory mechanism. Viewed through this lens, formation of RNA‐protein granules is a regulatory response, whose functions include translational regulation.Support or Funding InformationThe molecular and cellular biology training grant T32 GM007183