bluuebunny/crossref_metadata_2025_split huggingface dataset:
QscR regulates expression of the formate dehydrogenase genes in Methylobacterium extorquens AM1 (946.9)
Authors: Valdez Rachael , Skovran Elizabeth 
Published: June 2021

Methylobacterium extorquens is a model organism for understanding methylotrophic growth. It is of interest as a platform for production of value added chemicals, such as biofuel and bioplastics, from methanol which is inexpensive and readily available. During methanol growth, methanol is first oxidized to formate by methanol dehydrogenase and the formaldehyde activating enzyme. The cell then faces an important choice. Formate can be oxidized to CO2 producing cellular reducing power, or be converted to methylene‐H4F which is assimilated into biomass. Four formate dehydrogenases are known to exist in M. extorquens, though how these enzymes are regulated is unknown. QscR plays an important role during methylotrophic growth by activating expression of the Serine Cycle genes, which constitute the main carbon assimilation pathway during methylotrophic growth. QscR directly senses formyl‐H4F, an intermediate produced after formate but before the Serine Cycle. Microarray data suggest that QscR may also regulate the expression of the formate dehydrogenases genes. To assess in vivo regulation of formate dehydrogenase expression, fluorescent transcriptional reporter fusions were created. QscR was also purified and promoter binding was assessed. At least two of four formate dehydrogenase promoters were shifted by QscR. We also show that QscR derepresses its own transcription in the presence of formate suggesting that a feedback loop exists, which results in derepression of qscR expression when formate builds up and repression of qscR expression as levels of formate decrease and formyl‐H4F increases. This co‐metabolite sensing by QscR would allow the cell to coordinate carbon assimilation and dissimilation. A deeper understanding of the regulation at the formate branch point will be essential in engineering M. extorquens to balance energy and reducing power generation with carbon assimilation into value added chemicals.Grant Funding Source: Supported by: San Jose State University Research, Scholarship and Creative Activity Grant
Assessing the orientations of lipoprotein Pal in the outer membrane of Escherichia coli (948.4)
Authors: MacPherson Victoria , Shaw Juliana , Pichichero Michael , Michel Lea 
Published: June 2021

Pal is a well‐characterized peptidoglycan‐associated lipoprotein found in the periplasm of Escherichia coli (E. coli). Although the biological function of Pal is not understood, the protein has been shown to interact with Tol B, Lpp, and several other periplasmic proteins. We recently discovered that Pal’s homologue, P6 of nontypable Haemophilus Influenzae (NTHi), is inserted into the outer membrane in two orientations—one facing in toward the periplasm and one facing out toward the extracellular space. One other lipoprotein, Lpp of E. coli, was also shown to exhibit two orientations in the outer membrane. In an effort to collect data in support of our hypothesis that lipoprotein dual orientation is a more widespread phenomenon, we utilized confocal microscopy and flow cytometry to assess the localization and orientation(s) of Pal in E. coli. Our results demonstrate that Pal is indeed present on the surface of E. coli, suggesting that Pal is also a dual oriented lipoprotein.Grant Funding Source: Supported by the Camille and Henry Dreyfus Special Grant Program in the Chemical Sciences.
The structure of a tail‐anchor membrane protein‐binding complex reveals the regulation of Get3 by Get4 (950.4)
Authors: Gristick Harry , Rao Meera , Chartron Justin , Rome Mike , Shan Shu‐ou , Clemons William 
Published: June 2021

Tail‐anchored (TA) membrane proteins are targeted to the endoplasmic reticulum via the post‐translational GET pathway. A critical step in this essential pathway is the transfer of TA substrates from the co‐chaperone Sgt2 to the Get3 ATPase, mediated by the hetero‐tetrameric complex Get4/Get5 (Get4/5). However, a major outstanding question is how Get4 binds to Get3 and regulates its activity. Here we report a 5.4 Å crystal structure of an ATP‐bound Get3‐Get4/5 complex from Saccharomyces cerevisiae (Sc), the largest pathway components solved to date. Our structure reveals the role of nucleotide in complex formation and the presence of two functionally distinct binding interfaces. Mutational analysis confirms that one interface is required for the specificity of binding, while the other interface is involved in the regulation of Get3 ATPase activity. Additional functional assays demonstrate that Get4/5‐mediated regulation of ATP hydrolysis by Get3 is essential for efficient TA protein targeting. Our work also supports a model for Get3/TA complex formation in which two opposing Get3 dimers are bridged by a single Get4/5 hetero‐tetramer. Finally, we present a 2.8 Å crystal structure of an apo ScGet3‐Get4/5 complex that provides evidence for a rapid initial binding state mediated by electrostatics. This work illustrates how Get4/5 regulates Get3, priming it for TA loading, a critical step in this important pathway.Grant Funding Source: NIH
Molecular mechanisms of substrate degradation by the 26S proteasome revealed by CryoEM (952.6)
Authors: Matyskiela Mary , Lander Gabriel , Martin Andreas 
Published: June 2021

The 26S proteasome is the major eukaryotic ATP‐dependent protease, responsible for regulating the proteome through degradation of ubiquitin‐tagged substrates. The proteasome regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains, and translocates them into the associated peptidase after unfolding. Despite recent insights into the architecture of the regulatory particle, little is known about the structural requirements and mechanisms for substrate processing. Here, we present the first structure of the 26S proteasome during substrate degradation and show that it switches from a pre‐engaged to a translocation‐competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase, and a spiral orientation of pore loops that suggests a rapid progression of ATP‐hydrolysis events around the ring. Importantly, Rpn11 moves from an occluded position to directly above the central pore, facilitating substrate deubiquitination concomitant with translocation.Grant Funding Source: Supported by NIH grant R01‐GM094497‐01A1, ACS grant 121453‐PF‐11‐178‐01‐TBE
Membrane fluidity and lipoperoxidation of placental mitochondria in diabetic rats (953.2)
Authors: Figueroa‐García María del Consuelo , Cortés‐Rojo Christian , Saavedra‐Molina Alfredo , Mejía‐Zepeda Ricardo 
Published: June 2021

Oxidative damage on biological molecules has been proposed as a major cause of alterations observed during diabetes. In this study, using the excimer forming monitor dipyrenylpropane (DPyP), we measured membrane fluidity of placental mitochondria in diabetic rats with 19 days of gestation and its respective normoglycemic controls. A significant increment (p < 0.001) of mitochondrial membrane fluidity was found in diabetic rats, along with MDA increment. Physicochemical parameters had changes, i.e. lower activation energies in gel (23.0 ± 5.0 vs. 18.2 ± 4.0 kJ/mol) and liquid‐crystalline (13.9 ± 1.3 vs. 12.0 ± 0.4 kJ/mol) phases in the diabetic group (p < 0.05); the transition temperature was lower in diabetic (37.8 ± 1.6 vs. 30 ± 2.0 °C), p < 0.05. There were several changes in fatty acid composition. These results provide further evidence that oxidative stress may play a role in the increment of mitochondrial membrane fluidity during pregnancy in diabetic rats despite the physical effects of lipoperoxides in membranes.Acknowledgements: CONACYT postdoctoral Fellowship to MCFG; financial support from DGAPA‐PAPIIT UNAM to RMZ; financial support from CIC‐UMSNH (2.16 to ASM) and CONACYT (169093 to ASM).
Comparison of different techniques for monitoring of mammalian cell respiration (960.4)
Authors: Kondrashina Alina , Dmitriev Ruslan , Papkovsky Dmitri 
Published: June 2021

Cell and tissue oxygenation in vivo are maintained within narrow physiological range and their deviations from the norm lead to pathological states such as ischemia, cancer and inflammation. The selection of the analytical method for O2 monitoring is dictated by the required experimental design, sensitivity, throughput and measurement task. Comparison of different cell respiration techniques is important for their standardisation and benchmarking. Using intracellular and extracellular phosphorescent oxygen probes, we measured respiration of MEF cells in hermetically sealed quartz cuvettes, 96 well plates sealed with mineral oil on top of the samples (partial seal), and local oxygenation of resting cells in an open plate. In the latter cell oxygenation levels were shown to be determined by their respiration activity and ambient O2, whereas in quartz cuvettes absolute oxygen consumption rates (OCR) were directly proportional to cell numbers. Measurements of oxygen concentrations were performed with adherent and suspension, resting and stimulated cells on both platforms in parallel employing intracellular oxygen sensitive probe. We found a sigmoidal relationship between OCR and oxygenation with a linear correlation in the range 20‐200 μM O2 and leveling off at both high and low cell numbers which indicates changes in mitochondrial oxidative phosphorylation.Grant Funding Source: Supported by EU Grant Agreement number PITN‐GA‐2010‐264772 (ITN CHEBANA)
Cytokinesis requires supervillin‐mediated binding to myosin II and synergism with anillin (962.1)
Authors: Luna Elizabeth , Smith Tara , Fridy Peter , Li Yinyin , Basil Shruti , Arjun Sneha , Friesen Ryan , Leszyk John , Chait Brian , Rout Michael 
Published: June 2021

Cytokinesis requires coordination of cortical myosin II activation with central spindle alignment. Supervillin and anillin are two actin‐ and myosin II‐associated vertebrate proteins implicated in maintenance of the cytokinetic furrow in early cytokinesis, The nature of their cross‐talk is unknown. Point mutations within an evolutionarily conserved sequence in supervillin, residues 99 ‐ 153, eliminate binding to the nonmuscle myosin II heavy chain (MHC), confer dominant‐negative inhibition of cytokinesis, and abrogate the ability of full‐length supervillin to rescue RNAi‐mediated depletion of supervillin. While an observed ~2‐fold increase of anillin in supervillin‐knockdown cells does not eliminate cytokinetic failure, knockdown of both proteins together yields more than additive increases in numbers of multinucleated cells, as compared with depletion of either protein alone. Localizations of supervillin, anillin, MHC (total myosin), and phosphorylated regulatory myosin II light chain (pMRLC, activated myosin) in dividing cells suggest that supervillin and anillin synergize to confine MHC to the furrow. Anillin alone is sufficient to maintain normal levels of pMRLC at the furrow in late anaphase, but supervillin is required for normal pMRLC localization in early anaphase and around the cytokinetic bridge during daughter cell separation. Protein affinity isolations of GFP‐tagged supervillin and anillin indicate that these proteins do not significantly associate with each other although myosin II, actin and other cytoskeletal proteins co‐purify with each bait protein. None of these proteins co‐isolated with GFP alone. Stark differences between specifically co‐isolating kinases, phosphatases, and other signaling proteins reinforce the conclusion that supervillin and anillin regulate cytokinesis through separate pathways.Grant Funding Source: Supported by the National Institutes of Health and the University of Massachusetts Medical School.
A protein crystallography and small molecule informatics‐based discovery engine for the development of selective chemical probes for in vivo investigations of protein kinases (968.3)
Authors: Grum‐Tokars Valerie , Roy Saktimayee , Minasov George , Anderson Wayne , Watterson D. 
Published: June 2021

There is a critical need for selective serine‐threonine protein kinase (S/T PK) inhibitors appropriate for in vivo studies. We used a discovery engine based on high resolution co‐crystallography of PKs in complex with ligands and robust databases for small molecules with documented in vivo properties and potential for good ADMET property development (Chico et al., 2009a, 2009b). As a proof‐of‐concept, we targeted the active site of the stress activated p38α MAPK, a well‐studied target that has been intractable for PK specificity when targeting the active site. Further, we focused on the especially difficult challenge of p38α MAPK involvement in CNS pathology progression where it is activated by diverse stressors in both glia and neurons as part of a synaptic dysfunction cycle. Novel, highly selective, CNS penetrant, active site bound, p38α MAPK inhibitors were developed and validated by large scale kinome and pharmacological screens, cellular target engagement, and in vivo proof of efficacy (Watterson et al., 2013).Grant Funding Source: NIH grants R01 NS056051, R01 AG031311, U01 AG043415
Characterization of glycosyltransferases in the biosynthetic pathway of capsular polysaccharide A (970.4)
Authors: Sharma Sunita , Troutman Jerry 
Published: June 2021

Capsular Polysaccharide A (CPSA) is a polymer of a tetrasaccharide subunit found on the surface of the symbiotic gut bacteria Bacteroides fragilis. It has been suggested that CPSA is vital for maintaining a normal balance between the various types of immune cells in a healthy mammalian host. If this balance is disturbed, various autoimmune disorders may develop in the human body. Our laboratory is applying an in vitro method to enzymatically synthesize CPSA, which is assembled on a bactoprenyl diphosphate scaffold one sugar at a time. The initiating sugar transferase, WcfS has been previously identified and characterized. Utilizing reverse phase HPLC, and fluorescent bactoprenyl monophosphate analogues we will describe the functional characterization of the remaining glycosyltransferases involved in CPSA biosynthesis, WcfN, WcfP and WcfQ.Grant Funding Source: NIH
Towards the functional characterization and inhibition of hydrolase enzymes in mycobacteria (972.5)
Authors: Patel Hiren , Seeliger Jessica 
Published: June 2021

The emergence of multiple and extremely drug‐resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) in humans, threatens the usefulness of our current therapeutic arsenal. As a result, there is an increasing need to identify novel mycobacterial targets that may lead to shorter, more effective treatment regimens. One promising group of potential targets is the serine hydrolases, which have diverse activities, but use a conserved nucleophilic serine residue for catalysis. In Mtb, many SHs have essential functions for Mtb survival, which makes the SH family a promising candidate for drug screening in mycobacteria. Using activity‐based probe profiling, the SH proteomes were visualized in three mycobacterial species that serve as models for virulent Mtb. Distinct populations of proteins were detect in the cell envelope versus the cytosol and between species. Importantly, mass spectrometry analysis of the SH proteome from attenuated Mtb identified known SHs, bioinformatically predicted SHs, and unannotated proteins as SHs. Furthermore, pre‐treatment of attenuated Mtb lysates with small‐molecule inhibitors resulted in concentration‐dependent inhibition of select enzymes. In conclusion, these data suggests that interspecies differences in the mycobacterial SH proteome can aid in understanding Mtb pathogenesis and that using activity‐based probe profiling will help identify selective inhibitors that have potential antimicrobial activity or can be used for characterizing newly annotated SHs. Therefore in the future, we will pursue small‐molecule screens to identify inhibitors that will aid in the functional characterization of these newly annotated SHs. Grant Funding Source: Supported by NIGMS T32‐GM008444, NIGMS T32‐GM092714, and the American Lung Association
Compartmentation of azelate and glutarate metabolism in rat liver (982.5)
Authors: Jin Zhicheng , Tomcik Kristyen , Bian Fang , Brunengraber Henri , Zhang Guofang 
Published: June 2021

Azelate (C9) and glutarate (C5) are odd‐chain dicarboxylates. Azelate, derived from the oxidation of oleate, is an inhibitor of tyrosinase and thioredoxin reductase. The compartmentation of azelate metabolism and its final metabolic fates are not known. We investigated the metabolism of azelate in rat liver using a combination of metabolomics and mass isotopomer analysis. Isolated rat livers were perfused with 2 mM (un)labeled azelate or glutarate ([13C9]azelate or [13C5]glutarate) for 2 h in recirculating mode. Perfusate and liver were analyzed by GC‐MS and LC‐MS/MS. The data showed that: (i) azelate oxidation starts in peroxisomes, and ends in mitochondria; (ii) glutarate oxidation occurs only in mitochondria; (iii) azelate forms pimeloyl‐CoA and glutaryl‐CoA, but not malonyl‐CoA, (iv) an unknown reaction removes one carbon from azelaoyl‐CoA forming octanedioyl‐CoA, then hexanedioyl‐CoA, (v) the metabolism of glutarate to acetyl‐CoA via 3‐hydroxyglutaryl‐CoA, crotonyl‐CoA and S‐3‐hydroxybutyryl‐CoA is confirmed.Grant Funding Source: NIH R33DK07029 and Cleveland Mt. Sinai Health Care Foundation
Evaluating the effect of formalin fixation on mass spectrometry‐based proteomic profiling (984.1)
Authors: Atwood James , Neumann Drexel , Dammer Eric , Duong Duc , Dunn Chelsea , Seyfried Nicholas 
Published: June 2021

Intro: Formalin fixation is the universal standard for preserving tissue samples. However, due to the nature of the formalin fixation process, protein extraction and recovery is typically compromised when compared to proteomic analysis of fresh tissues. Herein, we performed a comparative proteomic analysis between matched fresh and formalin fixed brain tissue samples in order to evaluate the degree of proteome degradation and to establish and optimized protocol for protein extraction from formalin fixed samples.Method: Fresh and free floating formalin fixed brain tissues were homogenized under non‐denaturing and denaturing conditions. Protein recoveries were quantified and the protein mixture was enzymatically digested. Resulting peptides were analyzed by quantitative LC‐MS/MS to compare total proteome coverage between tissue types and preparation methods.Results: While total protein recovery was comparable across both tissue types the homogenization buffer affected yields dramatically. Proteomic analysis revealed a significant decrease in total proteome coverage in the formalin fixed tissues with proteins of known high abundance being most affected.
Genesis of antibiotic resistance VI: monitoring of water quality for antibiotics, its metabolic breakdown products in the Texas Clean Rivers Program by the Texas Commission on Environmental Quality and also by the Texas Water Development Board (986.6)
Authors: Kannan Subburaj , Castillo Jessica , Estrada Courtney , Subburaj Prabavathy 
Published: June 2021

Established in 1991, the Texas Clean Rivers Program (CRP) is a non‐profit regulatory program for managing and/or monitoring water quality issues at the watershed level, within the river basins in Texas. A systematic data query for antibiotics, their metabolic breakdown products (Chemical intermediates, Parent ‐ Daughter Transition Compounds), and antibiotic resistant bacterial pathogens on surface water quality monitoring database [http://www.tceq.texas.gov/waterquality/clean‐rivers/data/samplequery.html], collected under a TCEQ‐approved quality assurance, conforms to the provisions of NELAP (National Environmental Laboratory Accreditation Program, July 2008). Storet queries for “Antibiotics” “Metabolic breakdown products of Antibiotics” , “Antibiotic Resistant bacterial pathogens”, and Antibiotic Resistant bacterial pathogens in fish tissues”, in the data base: http://www.tceq.texas.gov/waterquality/clean‐rivers/data/storet.html for the period of 2000 ‐ 2012 shows no data on file for said queries. Similar data queries in the TWDB database at http://www.twdb.texas.gov/groundwater/data/index.asp shows no data on file. The implication on lack information or scant information on said quires, and also most recent theoretical projections on “Antibiotic Resistance in the United States in 2013 by Center for Disease Control and Prevention is a serious public health concerns. Lack of data analysis from the Water Quality data supporting the CDC&P claims shall be presented in the Experimental Biology of 2014. Grant Funding Source: Professional Developement Funds from SWTJC, Eagle Pass
Molecular taxis: impact of candidate genes on epithelial sodium channel function (997.7)
Authors: Hall Jeffrey , Booth Rachell 
Published: June 2021

The epithelial sodium channel (ENaC) is a heterotrimeric transmembrane protein located in the apical membrane of the renal epithelium. This highly selective ion channel plays an important role in the homeostasis of both electrolyte and extracellular fluid levels in mammalian systems. While a number of molecular pathways responsible for regulation of ENaC‐such as those through SGK1‐dependent activation, Nedd‐4 ubiquitination preceding proteosomal degradation, and others‐the entirety of the ENaC regulatory pathway is not known. Our lab has expressed homotrimeric αENaC channels in yeast mutants taken from a yeast deletion library, each chosen due to general roles in endosomal trafficking and membrane localization. In order to assess the impact of genomic mutations in the presence of exogenous sodium chloride, a serial dilution growth assay was performed and mutants exhibiting phenotypic variations from wild type were identified. Specifically, three deletion strains were observed to have significant differences in growth patterns, as compared to control phenotypes. The HRD1 and SSM4 genes encode ubiquitin‐protein ligases responsible for degradation of misfolded proteins associated with the endoplasmic reticulum (ER). The SCJ1 gene codes for a chaperone protein involved in mediating maturation of proteins in the ER. Further characterization of candidate gene interactions with ENaC will provide greater insight into the regulation of an important player in sodium homeostasis.Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmental Grant AI‐0045