bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allosteric regulation and substrate specificity of the proteasome deubiquitinase Rpn11 (936.2)
Authors: Worden Evan , Martin Andreas 
Published: June 2021

The 26S proteasome is a multi‐protein complex responsible for ubiquitin‐mediated protein degradation in eukaryotic cells. Rpn11, the only essential proteasome deubiquitinase, removes ubiquitin chains from protein substrates during degradation, however very little is known about the regulation and intrinsic ubiquitin cleavage specificity of the enzyme. We utilized a heterologous expression system for the proteasome lid subcomplex, mutational analyses and in‐vitro deubiquitination assays to study the activity of Rpn11 in different assembly intermediates of the proteasome. We found that Rpn11 shows no preference for K48 or K63 linked di‐ubiquitin, indicating that it is promiscuous in substrate cleavage specificity. Furthermore, our studies have shown that Rpn5 inhibits Rpn11 in the lid complex and that this inhibition is relieved by incorporation of the lid into the proteasome holoenzyme, or by truncation of the Rpn5 N‐terminus. These observations support a model where the N‐terminus of Rpn5 blocks the Rpn11 active site in the isolated lid, but swings away from the enzyme to make contacts with the base and core particle upon formation of the proteasome. This allosteric regulation by Rpn5 may prevent spurious dubiquitination by Rpn11 in the isolated lid and thus ensure that the enzyme only acts on substrates committed to proteasomal degradation.Grant Funding Source: Supported by National Science Foundation Graduate Research Student Fellowship
The Escherichia coli Tol‐Pal complex is essential for the positioning of proteins at the cell poles (948.3)
Authors: Rajendran Madhusudan , Santos Thiago , Weibel Douglas 
Published: June 2021

Organisms regulate essential biological processes, including growth, division, motility, and virulence, by controlling the subcellular localization of proteins and other biomolecules. Even though bacterial cells lack a complex internal system of endomembrane, they use numerous mechanisms to establish, maintain and replicate their subcellular organization. Particularly, the bacterial poles are home to numerous cytoplasmic and membrane proteins. However, the molecular mechanisms regulating the non‐uniform localization of polar proteins are largely unknown. Here we show that the Tol−Pal complex_a conserved component that maintains cell wall integrity in Gram‐negative bacteria_plays a key role in the polar localization of chemoreceptors in Escherichia coli. Mutants lacking the Tol−Pal complex and expressing fluorescently labeled chemoreceptors showed characteristically mis‐localized (i.e., non‐polar) chemoreceptors dispersed along the length of the cells. Cell motility and chemotaxis assays showed that the mutants presented motility and chemosensory defects that were not observed in the wild‐type strain. My long term goal is to investigate the role of the Tol−Pal complex in a diverse group of bacteria, with a focus on bio‐medically relevant organisms.Grant Funding Source: Supported by National Institute of Health (1DP2OD008735‐01)
Generation of a new monoclonal antibody against zebrafish Insm1a (951.2)
Authors: Hoerter Jacob , Forbes‐Osborne Marie , Morris Ann 
Published: June 2021

Zebrafish have the ability to regenerate many tissues including those of the central nervous system (CNS). The retina, the photosensitive tissue in the back of the eye, is an outgrowth of the CNS and has been shown to be capable of of regeneration after damage. The transcription factor Insm1 is expressed in the developing pancreatic and nervous system, neurogenic regions of the adult brain, and tumors of neuroendocrine origin. Additionally, in a model of chronic rod photoreceptor cell (PRC) degeneration, the progenitor cells involved in regeneration of rod PRCs express insm1a. Knock down of the retinally expressed zebrafish co‐ortholog of Insm1, insm1a, during embryonic development, resulted in delayed maturation of cone PRCs, and a loss of rod PRC differentiation.Additional experiments are required to determine the mechanism by which Insm1 affects rod PRC differentiation during development, as well as its role in regeneration. Because Insm1 is a transcriptional regulator, it is important to know what downstream genes are regulated by Insm1. For these experiments, an antibody specific for zebrafish Insm1a is necessary. However, no suitable antibody is currently available. Thus, we decided to produce a monoclonal antibody against the entire coding region of insm1a. After cloning the protein coding region of insm1a downstream of a 6‐histidine tag, we used an inducible bacterial expression system to produce tagged Insm1a in vitro. The protein was successfully purified by affinity chromatography. After sequencing, the protein will be used as the immunogen in a monoclonal antibody screen. A purified antibody that specifically recognizes Insm1a will allow us to perform chromatin immunoprecipitation experiments to identify which promoters are regulated by insm1a. This information will help us to place Insm1 within a known transcriptional regulatory network and will provide information about the mechanisms by which Insm1 transcriptional regulation influences retinal development.
Identification of E6AP/UBE3A as a ubiquitin‐protein ligase that catalyzes encephalomyocarditis virus 3C protease ubiquitylation (952.1)
Authors: Notarianni Tara , Carmody Marybeth , Kirwin Katherine , Lawson T. 
Published: June 2021

The 3C proteases of encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) are rapidly degraded by the ubiquitin‐26S protease system, which limits the 3C protease concentration that develops in infected cells. We have discovered that at least one UbcH7‐dependent ubiquitylation pathway is important for EMCV replication success determination. To identify the UbcH7‐dependent E3 ubiquitin‐protein ligase(s) involved in tagging the 3C proteases for destruction, we developed a protein purification scheme that begins with mouse cell lysate and ends with a ubiquitin‐UbcH7 affinity column. Analysis by mass spectroscopy of the proteins eluted from this column revealed the presence of four E3s, one of which is the HECT E3 E6AP/UBE3. The ability of this E3 to recognize the EMCV 3C protease as a substrate was confirmed using reconstituted mixtures containing purified E6AP. The major product of these reactions was monoubiquitylated 3C protease rather than polyubiquitylated conjugates. Whether this accurately reflects E6AP action in vivo remains to be determined, but it is possible that E6AP must function with other E3s to catalyze 3C protease polyubiquitylation in infected cells. Either UbcH7 or UbcH5 support E6AP‐catalyzed EMCV 3C protease ubiquitylation, with a slight kinetic preference for UbcH7. While the HAV 3C protease is also ubiquitylated by a UbcH7‐dependent E3, our results show that this E3 is not E6AP.Grant Funding Source: Supported by NIH Award 1R15AI099134‐01.
β‐Actin is regulated by endothelin 1 and is not a consistent endogenous control in aortic smooth muscle cells from leptin‐deficient mice (955.1)
Authors: Carmo Luciana , Andrade Maria Claudina , Liberman Marcel 
Published: June 2021

β‐actin, a highly conserved gene/protein, which is usually not modulated by different stimuli, is routinely used to normalize target gene or protein expression in experimental biology, and so defined endogenous housekeeping gene. However, investigators showed that β‐actin expression could change during cellular growth and differentiation. We hypothesized that endothelin 1 (ET‐1), a cellular proliferative agonist, increase β‐actin expression in vascular smooth muscle cells (SMC). Primary aortic SMC passage 4‐6 isolated from obese leptin‐deficient (obob) and from C57BL/6 mice were incubated without (Cont) or with ET‐1 50nM (ET) for 0‐72h. Cell lysates were assessed for β‐actin expression by Western blotting. ObobET SMC increased β‐actin expression after 24h, 48h and 72h (1.68 ±0.06, 1.66 ± 0.05, 1.73 ± 0.06 respectively) normalized by GAPDH vs obobCont (1.01± 0.07), p<.05 n=6. However, C57ET did not change β‐actin expression (0.91 ±0.06, 1.04 ± 0.03, 0.99 ± 0.08) after 24h, 48h and 72h respectively vs. C57Cont (0.95 ± 0.04), p=NS n=6. We showed that ET‐1 modulates β‐actin expression only in obob SMC, but not in C57BL/6 SMC. In conclusion, β‐actin is not a consistent endogenous control in ET‐1‐stimulated SMC from obob mice. Grant Funding Source: Supported by FAPESP 2010/07455‐5
It takes two to tango: fine‐tuning of soluble guanylate cyclase activity via protein‐protein interactions and conformational changes (969.3)
Authors: Garcin Elsa , Seeger Franziska , Quintyn Royston , Tanimoto Akiko , Williams Garrett , Tsutakawa Susan , Wysocki Vicki , Tainer John 
Published: June 2021

Nitric oxide (NO) regulates many biological processes, including vasodilation, neurotransmission, and immune response. The primary receptor for NO is heterodimeric soluble guanylate cyclase (sGC), which is a validated target for therapeutic intervention in pulmonary arterial hypertension.We have solved the first x‐ray structure of wild‐type heterodimeric catalytic domains from human sGC, revealing key structural determinants for heterodimerization and conformational changes needed for catalysis. Using mass spectrometry, we determined dissociation constants for heterodimeric and homodimeric catalytic domains. Surprisingly, activity measurements showed that isolated catalytic domains are far less active than full‐length sGC.Together, our results argue that other sGC domains are crucial to promote sGC heterodimerization in vivo. We further propose an exquisite regulation mechanism whereby the catalytic activity of sGC is finely tuned by protein:protein interactions that either promote or inhibit catalytic activity. Importantly, our structural studies provide a framework to design novel sGC activators targeted at the catalytic domain.Grant Funding Source: Supported by American Heart Association
Dynamics of the general secretory system viewed in near‐native conditions via atomic force microscopy (993.1)
Authors: King Gavin , Sanganna Gari Raghavendar Reddy , Frey Nathan , Mao Chungfeng , Randall Linda 
Published: June 2021

In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein transfer across and into membranes. Further, it is known that SecA is the ATPase of the general secretory system and it binds SecYEG to perform translocation. In so doing, SecA makes large surface area contact with the unstructured cytoplasmic loops spanning transmembrane helices 6‐7 and 8‐9 of SecY. Despite their functional significance, measurements of flexible and disordered protein domains remain a significant experimental challenge. Recently, atomic force microscopy (AFM) has emerged as an important complementary tool in biophysics and is well suited for studying membrane protein dynamics in near‐native conditions (i.e., in a native lipid environment, at physiologically relevant temperature and ionic strength). We have studied purified SecYEG that was reconstituted into liposomes via AFM. After confirming activity, changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude and were attributed to the aforementioned loops of SecY. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. Finally, we have imaged SecA engaged on SecYEG and related the structural states observed to the activity of the translocase. This work provides a novel and near‐native vista of central components of the general secretory system.Grant Funding Source: This work was supported by the National Science Foundation CAREER Award 1054832 (to G. M. K.)
Intestinal divalent metal‐ion transporter‐1 is critical for intestinal iron absorption but not that of copper (996.3)
Authors: Shawki Ali , Anthony Sarah , Engevik Melinda , Niespodzany Eric , Worrell Roger , Mackenzie Bryan 
Published: June 2021

Divalent metal‐ion transporter‐1 (DMT1) is a widely expressed iron transporter serving iron absorption and erythroid iron uptake. DMT1 can also transport certain other transition metal ions; however, questions have arisen as to the role that DMT1 plays in the absorption of copper. We tested the hypothesis that intestinal DMT1 is essential for the absorption of Fe and Cu by examining (1) hematological variables and Fe status, (2) expression of Fe‐related genes (by using RT‐qPCR), and (3) radiotracer metal absorption in a mouse model lacking intestinal DMT1 (i.e. DMT1int/int, generated by crossing floxed DMT1 and villin‐Cre transgenic lines). The DMT1int/int mouse (at age ≍ 120 d) exhibited a profound hypochromic–microcytic anemia (hemoglobin concentration was 1.8 ± SD 0.5 g/dL in DMT1int/int mice cf. 14.6 ± 0.7 g/dL in wildtype, n = 7–9), and severely depleted serum Fe and liver nonheme Fe stores. mRNA expression of Cybrd1 (ferrireductase) was increased to (30 ± 3)‐fold (mean ± propagated SE, n = 3) in enterocytes of DMT1int/int mice cf. wildtype mice and that of the basolateral Fe exporter ferroportin, (2.7 ± 1.0)‐fold. Liver Hamp1 (hepcidin) expression was depressed by 97% ± 48%. Expression of the apical Cu transporter Ctr1 was increased to (2 ± 1)‐fold in enterocytes of DMT1int/int cf. wildtype mice, consistent with other models of iron deficiency. Parenteral Fe injection corrected the hematological and serum‐Fe variables, Fe stores, and gene expression levels in the DMT1int/int mouse. We measured the absorption of 59Fe and 64Cu in mice fed by intragastric gavage. Loss of intestinal DMT1 decreased the 0–4‐hour appearance of 59Fe in blood by 88% ± 24% cf. wildtype. Enterocyte 59Fe content at 4 h was 93% ± 42% lower in DMT1int/int cf. wildtype mice, indicating that the defect was at the level of apical uptake. Intestinal handling of 64Cu in the DMT1int/int mouse was no different from wildtype (P = 0.26). We conclude that DMT1 is critical for intestinal absorption of Fe but not Cu.Grant Funding Source: Supported by PHS Grant DK080047
Identification of accessory proteins necessary for epithelial sodium channel function (997.8)
Authors: Sterling Beau , Booth Rachell 
Published: June 2021

The epithelial sodium channel (ENaC) is a selective membrane ion‐channel responsible for the preservation of proper sodium concentration to maintain homeostasis of blood and epithelial fluids. Protein‐protein interactions are important for the regulation, assembly, and membrane insertion and/or retrieval of ENaC. Other proteins necessary for ENaC maturation and function may lead to increased or decreased ENaC activity. Strains from the Saccharomyces cerevisiae Deletion Library were selected on the basis that the deleted gene in that strain may likely interact with ENaC during formation or otherwise regulate its function after integration within the cell membrane. The αENaC gene was transformed into yeast and selected colonies were replica plated using a serial dilution pronging assay. Growth of the yeast expressing αENaC were altered relative to the control strains. Two deletion strains exhibited a significant difference in growth as compared to controls. These strains lacked a CUE1 (elevated growth) and a PEP12 (almost no growth) when expressing αENaC. The CUE1 gene codes for an ubiquitin‐binding endoplasmic reticulum (ER) membrane protein that recruits the ubiquitin‐conjugating enzyme Ubc7p to the ER where it functions in protein degradation. The deleted carboxyPEPtidase Y‐deficient protein, coded by PEP12, is a target membrane receptor (t‐SNARE) for vesicular intermediates that control entry of biosynthetic, endocytic, and retrograde trafficking.Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmen.tal Grant AI‐0045
Lactosylceramide promotes hypertrophy through ROS generation and activation of ERK1/2 in cardiomyoctes (999.1)
Authors: Mishra Sumita , Chatterjee Subroto 
Published: June 2021

Glycosphingolipids (GSLs) are integral components of the surface of essentially all types of cells and tissues including heart. Lactosylceramide (LacCer), a member of the neutral GSL family is synthesized from Glucosylceramide (GlcCer) by Lactosylceramide synthase, plays a pivotal role in the biosynthesis of complex GSLs and imparts several critical phenotypes. However, the role of GSLs in cardiac system is not known and physiological and pathophysiological consequences of myocardial sphingolipid species and their regional differences in myocardium remain obscure. We have determined the efficacy of a wide variety of GSL's to affect cardiac hypertrophy using cultured rat cardiomyocytes e.g. H9C2 cells and cultured primary neonatal rat cardiomyocytes. We employed multiple criteria to assess hypertrophy in our studies. These were: a) measurement of [3H] Leucine incorporation into protein, b) measurement of cell size and morphology and c) Real time quantitative mRNA expression assay for atrial natriuretic protein (ANP) and brain natriuretic peptide (BNP). Subsequently, mechanistic studies were performed to measure the involvement of various signaling transduction pathways.Our in vitro studies using cardiomyocytes reveal that LacCer dose‐dependently activated the increase in cell surface area, cell volume, protein content and expression of BNP/ANP mRNA. A comparative study using other GSL's did not induce hypertrophy. LacCer induced hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. LacCer also activated MAP kinases implicated in the cardiac hypertrophy signaling pathways. mRNA levels for c‐Jun and c‐Fos were increased by LacCer. Increases in c‐jun and c‐fos gene expression due to LacCer were abolished by inhibition of PKC as well as inhibition of extracellular signal‐regulated kinases (ERK) 1/2. We report here for the first time that LacCer induces hypertrophy in cardiomyocytes.
Channeling Your Inner Mutations (LB104)
Authors: Brinegar Kim , Davis Zachery , Davidson Katie , Fournier Ronald , Hoover Breeanna , Macias Nicholas , Maldonado Vivian , Galea‐Martinez Sandra , Grootenhuis Peter 
Published: June 2021

Approximately 70,000 people worldwide have the genetic disease cystic fibrosis (CF). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The protein consists of two membrane spanning domains (MSD1,2), two nucleotide binding domains (NBD1,2), and a regulatory domain (R). The domains ultimately form a chloride channel. When mutant CFTR is not present at the cell surface or is dysfunctional, altered chloride levels lead to the formation of sticky mucus in the lungs, which causes chronic infection and inflammation, and ultimately death. Although there are over 1,900 CFTR mutations known, the most common is the deletion of phenylalanine‐508(F508del), which leads to the misfolding of CFTR and reduced the amount of CFTR at the cell surface. A rare mutation is the G551D mutation, which leads to defective gating of the channel. By targeting two small molecule agents, researchers have made advances in treating the underlying cause of CF. The El Capitan High School SMART (Students Modeling a Research Topic) Team used 3D printing technology to create a model of the CFTR domains based on the crystal structure of related proteins.Grant Funding Source: Supported by grants from NIH‐CTSA and NIH‐SEPA.
The Role of Yeast Urea Amidolyase in Patients with Suppressed Immune Systems (LB105)
Authors: Cavins K. , Alphin D. , Braatz A. , Deleon‐Camacho F. , Emkay S. , Flees H. , Franitza A. , Gerwig A. , Groth E. , Granlund L. , Idell A. , Klug L. , Nakhla V. , Osberg Z. , Paniagua P. , Wallner J. , Rochon D. , Fangmann J. , St. Maurice Martin 
Published: June 2021

According to Rice University, 70% of people are affected by the infectious fungus Candida albicans. The immune system uses T and B cells to stop pathogens. People with suppressed immune systems, such as children with transplants, AIDs or cancer patients, lack functional T and B cells, so they rely on macrophages to destroy Candida. Candida can kill and exit macrophages due to an enzyme: urea amidolyase (UAL). While in the macrophage, Candida goes through a morphological switch from a sphere to a structure with hyphae due to an environment change. UAL converts urea to ammonia and CO2, creating an environment for hyphae to form, bursting the macrophage. The Greenfield SMART (Students Modeling A Research Topic) Team used 3D printing technology to model the four domains of UAL. The biotin carboxylase (BC) domain uses energy from ATP cleavage to attach CO2 to the swinging arm portion, or biotin carboxyl carrier protein (BCCP) domain. The BCCP domain swings across UAL, attaching CO2 to urea forming allophanate in the carboxyl transferase (CT) domain. Allophanate moves to the allophanate hydrolase (AH) domain, which hydrolyzes the allophanate into CO2 and ammonia. Increases in CO2 and ammonia cause hyphae to form, destroying macrophages and allowing Candida to spread. Since humans lack UAL, researchers could block UAL’s active sites to prevent Candida’s macrophage‐killing shape change, thus preventing systemic candidiasis without damaging human cells.Grant Funding Source: Supported by a grant from NIH‐CTSA.
Investigating cross‐linked peptides in bovine aortic lysyl oxidase (LB129)
Authors: Peng Kai , Greenaway Frederick 
Published: June 2021

Lysyl oxidase (LOX) isolated from bovine aorta contains five disulfide bonds and the lysyl tyrosyl quinone (LTQ) cross‐link. In attempting to locate the inhibitor‐modified amino acid residues resulting from addition of various suicide inhibitors, which may be in cross‐linked regions of the protein, we have been optimizing procedures for mass spectrometric identification of cross‐linked peptides produced during proteolysis of the enzyme. To covert the reactive quinone group of the LTQ to an invariant structure, LOX was reacted with phenylhydrazine before proteolysis. Recombinant human and purified bovine lysyl oxidase were first subjected to various proteolyses and mass spectrometry acquisition methods to determine optimal experimental conditions. Proteolytic procedures must produce peptides of manageable size and with termini that can be at least partially sequenced; chymotrypsin proved to be more suitable than trypsin. Optimizing conditions significantly increased the sequence coverage of LOX from 60% to 95%. Targeted MS/MS was used to identify cross‐link containing peptides using calculated m/z values and by utilizing the software packages xQuest/xProphet (ETH Zürich) and pLink (CAS) to find the cross‐link containing peptides.Grant Funding Source: Supported in part by Nuclea Biotechnologies