bluuebunny/crossref_metadata_2025_split huggingface dataset:
Understanding The Magic Touch (LB101)
Authors: Garb Ashley , Barbeau Delaney , Ihrig Daniel , Henke Noah , Opheim Allison , Hansen Brandon , Respalje Lindsay , Schroeder Mitchell , Garb Jodie , Weyer Andy , Zappia Katherine 
Published: June 2021

The Institute of Medicine estimates $635 billion dollars are spent annually on people with chronic pain conditions. One debilitating symptom of these conditions is hypersensitivity to touch, where daily activities can be painful. Few therapeutics to ameliorate mechanical hypersensitivity exist because the mammalian ion channels that sense touch are poorly understood. The mechanosensitive channel of large conductance (MscL) is an ion channel in Mycobacterium tuberculosis which allows bacteria to respond to mechanical stimuli by electrochemical response, regulating membrane ion flow. Research shows structural changes in MscL causes the protein to open, allowing ions into the cell. Key amino acids include hydrophobic residues I14 and V21, creating a constriction at the cytoplasmic surface. R98, K99, K100, E102 and E104 are possibly a ligand binding site, potentially participating in the ion conduction pathway. Residues at the N‐terminus of MscL, K3, F5, E7 and F8, may play a role in sensing membrane stretch. The Laconia SMART (Students Modeling A Research Topic) Team used 3D printing technology to model MscL. Understanding the structure‐function relationships of the MscL channel protein may lead to better comprehension of how human mechanosensitive ion channels, like the Transient Receptor Potential Ankyrin 1, work and lead to a cure for hypersensitivity to touch.Grant Funding Source: Supported by a grant from NIH‐CTSA.
Beta‐2 adrenergic receptor: a SMART team modeling project (LB106)
Authors: Ellingwood Lexy Ellingwood , Sanders Mitchell , Sanders Katie Sanders , Vinod Anjan , Wang James , Wang John , Link Tina , Zhu Mengyuan Zhu 
Published: June 2021

Asthma is a condition that affects millions. In response to allergens, the smooth muscles surrounding the lungs undergo bronchoconstriction which reduces the amount of air accessible. Bronchoconstriction can be countered by bronchodilation which results from the side effects of Beta‐2 adrenergic receptor (b2AR) antagonists. B2AR is a type of G protein coupled receptors (GPCRs) that bind to a drug like salmeterol and induce a conformational change that activates signal transduction in the cell that results in the relaxation of smooth muscle in the lungs. In 2012, Brian Kobilka and Bob Lefkowitz received a Nobel prize for their biochemical analysis and structural determination of b2AR. Kobilka used the T4 lysozyme to stabilize the protein and create a clearer view of the protein. The critical residues for agonist binding are Phe290, Asp113, Ser207, Ser203, Asn312 and Phe193. The Walton SMART Team (Students Modeling A Research Topic) modeled the b2AR using 3D printing technology demonstrating the interaction profile between the high affinity agonist BI‐167107 that stabilizes the active state of b2AR.Grant Funding Source: Supported by grants from the NIH‐SEPA and NIH‐CTSA.
Akt1 signaling in dopamine‐dependent behaviors (LB116)
Authors: Lo Yu‐Hsiang , Franke Thomas 
Published: June 2021

Background: One biochemical change acquired following induction of depression‐like behavior and exposure to drugs of abuse is decreased phosphorylation of the Akt1 serine‐threonine kinase.Methods: To examine the relevance of intact Akt signaling, we used Akt1‐mutant mice as genetic model. By combining biochemical, pharmacological, genetic and behavioral approaches, we examined if altered Akt signal transduction affects behavioral responses in experimental paradigms related to mesolimbic/nigrostriatal dopamine function.Results: Loss‐of‐function Akt1‐mutant mice exhibited a phenotype of increased susceptibility in the learned helplessness paradigm. In this experimental model for acquired behavioral depression, the exposure to inescapable electric foot shocks (IES) disrupts the subject’s escape to subsequent noxious stimuli. While no significant differences were observed between sham‐treated control mice depending on genotypes, IES‐treated Akt1‐mutant mice showed increased latencies of crossing and a higher number of escape failures when compared to IES‐treated wild‐type mice. In further support of the importance of intact Akt signaling in dopamine‐dependent behavioral responsiveness, Akt1‐mutant mice displayed enhanced responses to acute cocaine, diminished sensitization to chronic cocaine exposure and altered conditional place preference for cocaine.Western blotting and immunohistochemical analyses after IES training revealed genotype‐dependent changes in Akt phosphorylation and regulation of its substrates following IES training. These biochemical changes phenocopied differences in behavioral outputs when comparing wild‐type and Akt1‐mutant mice.Discussion: Several mechanisms have been proposed to explain increased excitability of DA neurons after drug exposure. One that is increasingly attractive involves dephosphorylation and inhibition of the serine/threonine kinase Akt. Reduced Akt kinase activity is frequently observed in psychiatric disorders with high comorbidity for substance use. By exploring Akt1‐dependent stress responses in the mesolimbic system using genetic models, we gain novel insights into the role of Akt signaling in regulating critical brain circuitry involved in motivation, decision‐making and reward.Grant Funding Source: Supported by NIH DA032280
Physical and Functional Interactions of the E. coli Monothiol Glutaredoxin GrxD Suggest a Role in FeS Apoprotein Maturation (LB132)
Authors: Saini Avneesh , Boutigny Sylvain , Baidoo Edward E.K. , Yeung Natasha , Keasling Jay D. , Butland Gareth 
Published: June 2021

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi‐protein systems such as Isc and Suf in E. coli. Additional accessory factors including the monothiol glutaredoxin, GrxD, are located outside of these defined systems. GrxD has been proposed to function as a shuttle protein acting to transfer FeS clusters between scaffold and carrier proteins, although specific functional role remain unknown. We have previously linked GrxD in FeS client protein interactions with the radical SAM enzyme MiaB. To address these different hypotheses, we conducted an extensive survey for physical interaction of GrxD with a comprehensive set of FeS cluster biosynthesis factors including cysteine desulfurase components (IscS, SufSE), FeS scaffold (IscU, SufBC2D) , carrier proteins (IscA SufA, ErpA, NfuA), HscA, HscB and various FeS apoproteins. We have employed various methods, including affinity co‐purification, analytical gel filtration and surface plasmon resonance (SPR) to obtain interaction data. To date we have observed no physical interaction between GrxD and any FeS biosynthesis factor except the SufBC2D scaffold and the SufA carrier protein. With SufA a reproducible but extremely weak association is observed by SPR alone (KD >> 700 µM). In contrast we have observed specific interactions between GrxD and two radical SAM enzymes, MiaB (KD =12 µM) and BioB (KD =34 µM). These data strongly indicate a role for GrxD in FeS protein maturation or repair, working downstream of the Suf systemGrant Funding Source: NIH award GM088196 to G.B
Biological Nitrile Reduction ‐ Structure and Mechanism (LB136)
Authors: Swairjo Manal , Kiani Maryam , Chikwana Vimbai , Lee Bobby , Iwata‐Reuyl Dirk 
Published: June 2021

The enzyme QueF catalyzes the NADPH‐dependent reduction of the nitrile group of 7‐cyano‐7‐deazaguanine (preQ0) to 7‐aminomethyl‐7‐deazaguanine (preQ1) in the biosynthesis pathway to the tRNA modified nucleoside Queuosine. The only nitrile reductase known in biology, QueF is a potential new candidate for industrial biocatalysis of nitrile transformations to amines. QueF belongs to the tunneling fold (T‐fold) structural superfamily built around a small ββααββ protein domain that oligomerizes to form a β2nαn barrel and facilitates interfacial catalysis of a variety of chemical reactions. In recent years, several crystal structures have been determined of unimodular and bimodular QueF enzymes, and of active site mutants and complexes with substrate and cofactor. Here we present crystal structures and biochemical and transient kinetic data that elucidate the mechanism of biological nitrile reduction and provide insight into the molecular evolution of T‐fold mediated interfacial catalysis. Structures of substrate‐bound QueF demonstrate that formation of a thioimide covalent adduct is a key step in the mechanism of reaction and that preQ0 binding, not thioimide formation, induces closure of the active site. A strictly conserved active site disulfide bridge suggests possible enzyme regulation by allosteric disulfide. We propose a mechanism for the QueF‐catalyzed reaction.Grant Funding Source: Sponsored by the National Institutes of Health and the National Science Foundation
Characterization of beta‐glucosidase from Physarum polycephalum (LB141)
Authors: Oh‐date Ayaka , Minami Yoshiko 
Published: June 2021

Physarum polycephalum, which is one of the true slime molds, has a unique life cycle that includes plasmodia, sclerotia, spores, and amoebae. Furthermore, the plasmodia cultured in liquid medium form a small lump called microplasmodia. The microplasmodia secrete a large amount of slime that includes the various proteins such as beta‐glucosidase: intracellular soluble, membrane‐bound, and secretory enzymes. Previously, we had reported that the membrane‐bound beta‐glucosidase BglM1, which has a molecular mass of 130 kDa, has a unique primary structure. The structure of BglM1 consists mainly of two regions: the N‐terminal region, which contains a typical glycosyl hydrolase family 3 domains and the C‐terminal region, which has a unique sequence that is unrelated to hydrolytic reactions. Notably, the C‐terminal region of BglM1 contains two calx‐beta motifs known as calcium binding sites, an RGD sequence, and a transmembrane region. In this study, we focus on the characteristics of a secretory enzyme that may be a membrane‐free type of BglM1.The molecular mass of the secretory enzyme purified in the presence of Ca ion and absence of Ca ion is 130 and 95 kDa, respectively. The 130kDa molecules react with antibodies that are specific for the BglM1 N‐terminal and C‐terminal regions. However, the 95kDa molecules are not detected by the C‐terminal antibody. These results indicate that in the absence of Ca ion, the 130kDa molecules are unstable and are degraded to 95kDa molecules. The kinetics data obtained from the 95 and 130kDa molecules showed that the calx region plays an important role on the stability and function of the enzyme. Further, to clarify the physiological functions of BglM1 with a calx region and of the secretory form, we assessed their mRNA expression during the Physarum cell cycle.
VPA inhibits myo‐inositol phosphate synthase activity by inhibition of Gsk3 (LB150)
Authors: Yu Wenxi , Greenberg Miriam 
Published: June 2021

Valproic acid (VPA) has been used for decades for the treatment of bipolar disorder. Yet, the therapeutic mechanism of VPA is not understood, hindering the development of more effective drugs. VPA causes inositol depletion in the human brain cells and in yeast cells. A similar degree of inositol depletion was observed in the yeast gsk3Δ mutant, in which the four glycogen synthase kinase 3 (GSK3) homologs were deleted including MCK1, MRK1, MDS1 and YGK3. VPA was also shown to inhibit Gsk3 in mammalian cells. These findings suggest that Gsk3 may play a role in inositol metabolism, and that VPA may cause inositol depletion by inhibiting Gsk3. The rate limiting enzyme in yeast inositol de novo synthesis is myo‐inositol phosphate synthase (MIPS). VPA treatment has been shown to cause a 50% decrease in MIPS activity. However, VPA did not inhibit MIPS in gsk3Δ cells, suggesting that VPA‐induced inositol depletion occurs by inhibition of Gsk3, leading to decreased MIPS activity. Of the four GSK3 homologs, we show that MCK1 is required for optimal inositol synthesis. mck1Δ is the only GSK3 single mutant that exhibited multiple features of inositol depletion, including inositol‐dependent growth, decreased intracellular inositol and decreased MIPS activity. VPA‐induced inhibition of MIPS was not observed in mck1Δ, indicating that Mck1 is required by VPA to inhibit MIPS. To verify that MCK1 is the sole GSK3 gene involved in regulating inositol metabolism, a triple mutant (mrk1Δmds1Δygk3Δ) was constructed. The triple mutant exhibited normal growth in inositol‐free media and increased intracellular inositol compared to WT, indicating that inositol depletion observed in gsk3Δ does not result from deletion of MRK1, MDS1 or YGK3. In conclusion, MCK1 is the yeast GSK3 homolog required for optimal inositol synthesis and VPA‐induced inhibition of MIPS is Mck1 dependent.Grant Funding Source: Supported by NIH‐DK081367 & Wayne State University Enhancement Award for Doctoral Student Travel
Characterization of the novel H82R mutation in CGI‐58 that causes Neutral Lipid Storage Disorder in humans (LB153)
Authors: Hassanien Sarah , McMahon Derek , Kurz Daniel , Shah Dharika , Dinh Anna , Brasaemle Dawn 
Published: June 2021

Comparative gene identification‐58 (CGI‐58) interacts with and co‐activates adipose triglyceride lipase (ATGL). The H82R mutation in human CGI‐58 causes a neutral lipid storage disorder (NLSD) characterized by ichthyosis and excessive triglyceride storage in many types of cells. We studied the comparable H84R mutation in mouse CGI‐58, and H84A mutated CGI‐58, to ask how CGI‐58 function is impaired. The ectopic expression of wild‐type (WT) CGI‐58 in human NLSD fibroblasts reduced excessive triglyceride storage to normal levels, whereas H84R CGI‐58 was significantly less effective. Additionally, neither H84R nor H84A CGI‐58 co‐activated ATGL in an in vitro triglyceride hydrolase assay. A co‐immunoprecipitation assay demonstrated that H84R and H84A CGI‐58 bound ATGL as well as WT CGI‐58. Immunofluorescence microscopy revealed that H84R and H84A CGI‐58 localized to ectopic perilipin 1A on lipid droplets of cultured NIH3T3CARΔ fibroblasts as well as WT CGI‐58. Moreover, the addition of forskolin and isobutylmethylxanthine to the cells triggered the dispersion of all three variants of CGI‐58 from the perilipin scaffold into the cytoplasm. Thus, H84R CGI‐58 shows appropriate subcellular localization in cells expressing perilipin 1A and binds to ATGL, yet does not co‐activate hydrolase activity. Future experiments will explore additional mechanisms for deficient CGI‐58 function.Grant Funding Source: Supported by NIH R01 DK054797.
Lipid raft abnormalities and subsequent protein trafficking effects in Niemann‐Pick type C1 (LB158)
Authors: Kuech Eva‐Maria , Shammas Hadeel , Maalouf Katia E. , von Koeckritz‐Blickwede Maren , Das Anibh M. , Naim Hassan Y. 
Published: June 2021

Many proteins and lipid components are organized within membrane microdomains, known as lipid rafts (LR). LR are cholesterol‐ and sphingolipid‐rich membranes resistant to non‐ionic detergents. In Niemann‐Pick type C1 (NPC1), a neurovisceral lysosomal storage disease, entrapment of cholesterol and sphingolipids occurs and may lead to alterations in LR composition. We investigate the LR status in fibroblasts derived from a patient with NPC1 and analyze the trafficking profiles of a typical LR‐associated membrane protein, dipeptidylpeptidase IV (DPPIV).Sucrose density gradients of Triton X‐100 cellular lysates revealed a substantial shift of the LR‐marker flotillin2 in NPC fibroblasts from the floating LR fractions to the detergent‐soluble membranes. Notably, the cell surface expression of DPPIV increased significantly by 2‐fold in NPC1 cells, while the overall protein level of DPPIV remained constant. FACS analysis corroborated the increase of DPPIV at the cell surface. This accumulation is the result of delayed lipid rafts‐dependent endocytosis in NPC1 cells as assessed by (i) a significant reduction in the level of internalized biotin‐labeled DPPIV and (ii) imaging studies revealing a reduced localization of DPPIV with EEA‐1 in NPC1 cells. Strikingly, N‐butyl‐deoxynojirimycin (NB‐DNJ), an inhibitor of glucosylceramide synthase, restored significantly LR levels and endocytosis of DPPIV. We conclude that altered composition or reduced levels of LR constitute a potential mechanism in the pathogenesis of NPC1 and NB‐DNJ treatment restores a balanced membrane lipid composition and the associated biochemical anomalies.
The Functional Significance of Phospho‐Rab GTPases (LB194)
Authors: Lepore Dante , Collins Ruth 
Published: June 2021

Intracellular membrane trafficking is an essential and conserved process throughout eukaryotes. To preserve organelle identity in a complex cellular system, control mechanisms must exist to oversee the production and targeting of intracellular vesicles. Key control elements are the Rab GTPases, which utilize a nucleotide‐bound cycle to act as master regulators for each discrete trafficking step. Several studies, including global proteomic screens, have identified multiple Rabs as phosphoproteins, but the functional consequence(s) of this modification are unknown. The Saccharomyces cerevisiae Rab GTPase Sec4p is negatively regulated by phosphorylation, but the molecular mechanisms and proteins responsible for this modification are unknown. Using Sec4p as a model for Rab phosphorylation and unique antibodies able to specifically detect modified Sec4p, we have shown that phosphorylation is tightly regulated in conjunction with cell cycle progression and spatial location within the cell. Currently, we are performing an overexpression screen to elucidate the kinase that modifies Sec4p as well as the biochemical pathway responsible for activating Sec4p phosphorylation. We hypothesize that Rab phosphorylation may be a novel regulatory mechanism through which additional cellular processes can impinge upon membrane trafficking.Grant Funding Source: National Institute of Health
Sec62 as a novel gating modulator for protein translocation and ion permeability of the mammalian Sec61 complex (LB196)
Authors: Schorr Stefan , Linxweiler Maximilian , Greiner Markus , Jung Martin , Cavalie Adolfo , Zimmermann Richard 
Published: June 2021

In mammalian cells the endoplasmic reticulum (ER) plays a key role during the biogenesis of secretory and membrane proteins and acts as a major regulator for intracellular Ca2+ signaling. Sec62, a component of the ER‐protein translocase, acts during post‐translational translocation of small precursor proteins and in ER‐Ca2+ leakage‐control. Our project aims to elucidate the molecular mechanisms how it regulates the translocation machinery concerning these two fundamental and seemingly opposing functions. We used siRNA mediated depletion of target proteins from human cells followed by functional assays for protein transport and live cell calcium imaging.Depletion of Sec62 in combination with proteasomal inhibition led to accumulation of the precursor of the ER resident protein ERj3 at the cytosolic face of the ER membrane. Control experiments using different siRNA as well as a plasmid rescue approach confirmed the specificity for Sec62. Follow‐up experiments revealed a pattern of dependencies upon translocon components which is characteristic for a post‐translational translocation pathway.Decrease of Sec62 level led to an increased ER calcium leakage that could be attributed to Sec61α. We observed that Sec62 binds the N‐terminus of Sec61α in a Ca2+ sensitive manner and identified a crucial putative EF‐hand in the C‐terminal part of Sec62.These experiments demonstrated for the first time that human Sec62 is important for the early steps of translocation of a large precursor protein indicating that Sec62 participation is not restricted to small proteins as suggested in previous studies.Additionally, Sec62 acts as a modulator, shifting the translocon from the open to the closed state after translocation is completed in order to maintain the ion impermeability of the ER membrane.Further experiments will allow us to determine the recently proposed role of Sec62 for various cancer entities.
The Effect of Maté on Enzymes Involved in the Recovery from Oxidative Stress (LB199)
Authors: Sadik Amina , Chang Theodore 
Published: June 2021

In comparing the effect of Maté to that of green tea, it has been shown that Maté has higher anti‐oxidative properties. We have shown that the extract of Yerba Maté, paraguariensis, leaves does indeed have strong anti‐oxidative properties through the evaluation of its scavenging activity and its ability of assist cardiomyocytes in their recovery from induced oxidative stress using both spectroscopy, a colorimetric assay and flow cytometry. In order to elucidate the mechanism behind this action, we quantified the enzymes known to be expressed naturally by cells under oxidative stress such as glutathione peroxidase, superoxide dismutase, and catalase. These enzymes were conjugated to antibodies and stains detectable via flow cytometry. Their expression was evaluated in presence and absence of oxidative stress. The obtained results indicated that Maté does not increase the expression of the genes coding for these enzyme but instead acts as a free radical scavenger, decreasing the amount of reactive oxidative species in the cellular environment, thereby decreasing the need for enzymes usually involved in the recovery from antioxidative stress. All the enzymes quantified showed a decrease in their expression in a dose‐dependent manner.
Upon discovering the inverse relationship of the activity of these enzymes to the concentration of Maté used to treat the cells, we concluded that Maté exerts it antioxidative capacity and therefore assists cardiomyocytes in their recovery from induced oxidative stress without or with very little involvement of the enzymes known to remove oxidative species from the cellular environment. These findings show that Maté is an effective antioxidant that prevents damage to the cardiovascular system.Grant Funding Source: Touro University Nevada
Identification of the functional binding domains of eukaryotic initiation factor eIF4G with 3’ Cap‐independent translation element of Barley Yellow Dwarf Virus (LB203)
Authors: Zhao Pei , Goss Dixie 
Published: June 2021

Different from canonical translation initiation, some plant viral RNAs lack a 5’cap (an unusual 7‐methyl guanosine linked by a 5’to 5’ triphosphate). They utilize a cap independent translation element (CITE) to efficiently start translation. Barley yellow dwarf virus (BYDV) has a translation element (BTE) located in its 3’UTR mRNA. BTE binds with eukaryotic initiation factor eIF4G (one subunit of heterodimer of eIF4F) and recruits other initiation factors to assemble the ribosome preinitiation complex for synthesis of viral proteins. BYDV is one of the world‐wide economically most important viruses causing severe crop loss. How BTE mediate BYDV translation is still unclear. By measuring fluorescence anisotropy of labeled RNA, here we report 3’BTE binds with a truncated fragment of eIF4G, eIF4G601‐1196 (contained amino acid residue from 601 to 1196). The equilibrium dissociation constants (Kd) of this binding is 39.5±3.5 nM. Adding eIF4E, the cap binding protein, can increase the binding ability of eIF4G601‐1196 with 3’BTE. eIF4G601‐1196•4E complex binds with 3’BTE 5.8 folds tighter than eIF4G601‐1196 alone, with the equilibrium dissociation constant 6.78±1.49nM. Further circular dichroism spectra (CD) and thermodynamic studies will characterize the binding of the eIF4G with 3’ BTE. These results suggest eIF4E plays an indirect role in translation initiation, even for an uncapped virus.Grant Funding Source: NSF MCB 1157632