bluuebunny/crossref_metadata_2025 huggingface dataset:
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
Intrinsically disordered N‐terminal domain of the steroid receptors: a promising target for endocrine‐based therapeutics (977.1)
Authors: Kumar Raj , Khan Shagufta
Published: June 2021

Organ/gene‐specific therapeutic targeting of steroid receptors (SRs) is presently restricted due, in part, to small molecule selective receptor modulators (SRMs) mainly targeting the structured pocket of the ligand binding domain to modulate co‐regulatory protein interactions with the AF2 and largely overlooking AF1 activity, located in the intrinsically disordered (ID) N‐terminal domain, which is a major contributor to cell/tissue and target gene specific actions of SRMs. It thus is axiomatic that attempts to more precisely control SR selectivity without understanding the actions of AF1 will be of limited success. A major challenge is the paucity of information on how and what kind of disorder‐order transition ID AF1 undergoes that leads to its functionally active folded conformation. In this study, we showed that the binding of glucocorticoid receptor’s ID AF1 to a target protein, as assessed by surface plasmon resonance (BIACORE), results into AF1 undergoing a disorder‐to‐order transition as determined by biophysical and proteomic techniques. Additionally, this induced folding of the AF1 is associated with enhancement of AF1‐mediated specific SR coactivators binding and subsequent transcriptional activity in vitro as determined by immunoprecipitation and promoter‐reporter assays. Targeting ID regions with small molecule drugs that can inhibit protein‐protein interactions at disorder‐to‐order transition is an emerging field with excellent promise; therefore, our results may provide additional SR selectivity needed to target cell‐tissue specific gene regulations in current endocrine‐based therapies.
Supramolecular interactions between peptides and a glycouril‐based molecular clip: a computational study (979.4)
Authors: Elsarrag Selma , Zachary Ralph
Published: June 2021

Selective recognition of amino acid residues in peptides by synthetic receptors has garnered much interest since an understanding of such processes may illuminate fundamental properties of biomolecular dynamics and assist in the rational design of therapeutic agents, sensors, and separation techniques. We have undertaken a computational study of a glycouril‐based molecular clip with the model peptides Ace‐Phe‐Nme and Ace‐Lys‐Nme with an eye towards understanding the relative binding strengths of resulting complexes, the particular intermolecular forces driving association, and the conformational changes induced in the peptide. Methods used include molecular dynamics, quantum mechanics at the density functional level of theory, and mixed quantum/molecular mechanics with explicit and implicit solvation models.
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
Bacterioferritin B (BfrB) and Ferredoxin (Bfd) associate to regulate iron release in the pathogenic bacteria Pseudomonas aeruginosa (LB107)
Authors: Harter Sydney , Einspahr Alana , Alzubbi Nadine , Dix Randy , Lovell Scott
Published: June 2021

Iron is an essential cofactor in respiration, nitrogen fixation, photosynthesis, and DNA synthesis and repair. Iron is necessary for the survival of pathogens in the host, thus iron storage proteins may be potential targets for antibiotics. Bacterioferritin (BfrB) is a 440 kDa iron‐storage protein that forms a 24‐mer assembly made up of 12 dimers and adopts a spherical structure with a hollow interior cavity. The individual subunits adopt a structure that consists of 4 α‐helicies and a short C‐ terminal helix. Each BfrB dimer binds a heme molecule for a total of 12 hemes in the assembly. During the storage process, reactive Fe2+ is oxidized to Fe3+ and the BfrB 24‐mer functions to catalyze the conversion of Fe2+ to Fe3+, which is stored in the central core of the assembly. Then to release iron, a total of 12 ferredoxin (Bfd) molecules bind to the dimers on the surface of BfrB, one Bfd molecule per BfrB dimer, to facilitate the release when needed by the cells. Fe3+ is reduced to soluble Fe2+ by the transfer of electrons from Bfd through the heme molecules of BfrB into the interior cavity. Understanding the interactions that enable iron homeostasis in bacteria is important, as iron storage proteins could be potential targets for the development of antibiotics. The Olathe North SMART Team (Students Modeling A Research Topic) researched and modeled BfrB‐Bfd using 3D printing technology.

Grant Funding Source: Supported by grants from NIH‐CTSA and NIH‐SEPA.
Fanconi anemia protein FANCM promotes replication traverse of DNA interstrand crosslinks (LB128)
Authors: Huang Jing , Liu Shuo , Bellani Marina , Ling Chen , Wang Yinsheng , Wang Weidong , Seidman Michael
Published: June 2021

Replication of the genome can be slowed or stalled by DNA interstrand crosslinks (ICLs), which are absolute blocks to helix unwinding. Current models propose that fork collisions, either from one (single fork model) or both (double fork model) sides of an ICL, initiate repair processes required for resumption of replication. To predict the relative probability of these two models in mammalian genomes, we have developed a DNA fiber based approach to visualize DNA replication in the vicinity of ICLs in mammalian genomes. We found evidence for both single and double fork models; however neither was the dominant pathway in mammalian genomes. Surprisingly, the most frequent replication patterns were consistent with replication fork traverse of an ICL, without lesion repair. The proteins of the Fanconi Anemia (FA) pathway are involved in both ICL repair and the cellular response to replication stress. We assessed the contribution of FA proteins to the replication patterns. The frequency of traverse events was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF protein complex. In contrast, deficiencies in the FA core proteins, which contribute to ICL repair, had no effect on the patterns. The results indicate that a FANCM translocase‐based mechanism, independent of the FA core complex, enable replication forks to continue past ICLs, leaving repair for a later time.

Grant Funding Source: NIH/INTRAMURAL PRIGRAM
Characterization of Bacillus AhpC_H1 as a novel type of thiol peroxidase (LB135)
Authors: Cha Mee‐Kyung , Kim Il‐Han
Published: June 2021

As a laboratory model organism Bacillus subtilis is used as the Gram‐positive equivalent of Escherichia coli, an extensively studied Gram‐negative rod. We found two types of thiol peroxidase (TPx) by searching a database of Bacillus substilis, which are homologous forms of E. coli AhpC. We named the two Bacillus TPxs as AhpC_H1 and AhpC_H2. In this present study, we characterized AhpC_H1 and AhpC_H2 purified from a recombinant E. coli. Structural studies of two AhpC homologs have shown that AhpC_H2 exists as a dimer form in oxidant condition like E. coli AhpC known as a typical 2‐Cys Prx, but most of AhpC_H1 are monomer form which is favored in oxidant conditions. AhpC_H1 has an additional Cys37 beside two conserved Cys47 and Cys166 unlike AhpC_H2. The presence of additional Cys37 was critical for monomeric form. Substitution of Cys37 impacts the monomeric pattern. Analyses of structural features on SDS PAGE gels of various Cys‐substituted proteins (C37S, C47S, C37/47S, C166S, C37/166S, C47/166S, and C37/47/166S) suggested a disulfide bond formed intramolecularly between Cys37 and Cys47. In addition to a chemical reducing reagent, thioredoxin system enables to catalyze the breakage of the disulfide bond. Its formation and breakage is shown to be important structural feature of the protein. The monomer and dimer transition in atypical AhpC_H1 displays a switch that could be involved in signaling in Bacillus subtilis. Based on these observations, we suggest that the AhpC_H1 is a new type of bacillus thiol peroxidase (TPx), and AhpC_H2 is a count part form of E. coli AhpC.

Grant Funding Source: This work was financially supported by NFR (2011‐0008913).
Sialidase and sialate O‐esterases promote cooperative mucus foraging by gut bacteria (LB145)
Authors: Lewis Warren , Robinson Lloyd , Lewis Amanda
Published: June 2021

Bacteria can gather nutrition from host secretions rich in carbohydrates (glycans). Sialic acids (Sias) are prominent outermost residues of glycans, and foraging on these molecules is a common theme among the microbiota of mammals. Many bacteria possess Sia catabolic pathways without encoding sialidase_they consume free Sia. Recent evidence suggests that of sialidases produced by other co‐habitant bacteria can liberate bound Sias to allow utilization by Sia consumers. Here we demonstrate cooperative Sia utilization by combinations of sialidases and free Sia consumers. We demonstrate that foraging on mucins by a free Sia consumer relies on the release of Sia by sialidase prior to breakdown by sialylate lyase. An additional obstacle to Sia utilization is presented by Sia O‐acetyl esters, which slow release of Sias by many bacterial sialidases. HPLC analysis shows that Sia O‐acetylation impedes Sia foraging by sialidase and a Sia consumer, and that recombinant sialate O‐esterases of gastrointestinal bacteria released both 7‐O‐ and 9‐O‐acetyl groups from sialic acid residues bound to glycan substrates, allowing efficient release by sialidase and subsequent consumption of Sias. Collectively, molecular and biochemical evidence supports cooperative mucus Sia foraging using on three enzymes contributed by members of the gut microbiota: sialate O‐esterase, sialidase, and sialate lyase.
Breaking the boundaries of the GPI transamidase complex (LB147)
Authors: G Gamage Dilani , Hendrickson Tamara
Published: June 2021

Breaking the boundaries of the GPI transamidase complex Dilani G. Gamage, and Tamara L. Hendrickson Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Glycosylphosphatidylinositol transamidase (GPI‐T) is a complicated, membrane‐bound, multi‐subunit enzyme that catalyzes an essential post‐translational modification.1‐3 This enzyme attaches GPI anchors to the C‐termini of various proteins that contain a proper GPI‐T signal sequence. Gpi8, Gaa1, Gpi16, Gpi17 and Gab1 are the five known subunits that may encompass the fungal GPI‐T; Gpi8 is the catalytic subunit but the functions of the other subunits remain largely unknown.5,6 In humans, different GPI‐T subunits are upregulated in different cancers, making GPI‐T a target for cancer research.7,8 However, in spite of the importance of this enzyme, little is known about how it assembles into an active enzyme complex, the stoichiometry of this complex, or the roles of the different components. Here we use soluble domains of the three core subunits (Gpi8, Gpi16 and Gaa1) to investigate the stoichiometry of the enzyme as well as to study the functions of each subunit. Results are shown for the homodimerization of the soluble domain of Gpi8 and the effect of N‐linked glycosylation on Gpi8 homodimerization. Co‐immunoprecipitation of the soluble domains of Gpi8:Gaa1, Gpi8:Gpi16 and Gpi8:Gpi16:Gaa1 discloses a minimal arrangement for these subunits in the absence of their transmembrane domains. Our results will help to elucidate the function and resolve the complexity of GPI‐T. To better understand how changes in expression affect GPI‐T activity, and as a model for this enzyme in cancer, we have also developed an in vivo strategy to monitor and quantify the effect of subunit overexpression on cell surface presentation of GPI‐anchored proteins in Saccharomyces cerevisiae.

Grant Funding Source: Wayne State University and the American Cancer Society.
Identification and characterization of autophagy‐related genes Atg8 in planarian (LB15)
Authors: Chuang Chao‐Yi , Tseng Ben‐Lyu , Liu Ang‐Yu , Huang Wei‐Pang , Chu Chia‐Ying
Published: June 2021

Autophagy is a highly conserved pathway from yeast to human, and a basic catabolic mechanism that degrades unnecessary or dysfunctional cellular components. Recently studies have shown that autophagy is involved in stem cell homeostatic control during aging, regeneration and tissue reprogramming. It has been shown that autophagy is required for self‐renewal and differentiation of adult human stem cells. While the function of autophagy in planarian stem cells and regeneration has not been clearly demonstrated yet.

To decipher if autophagy is required for planarian regeneration, we identified the homologs of several yeast Atg proteins from our transcriptome database of Dugesia japonica. We found three planarian homologs of yeast Atg8, and named them DjAtg8A, DjAtg8B, and DjAtg8C. In situ hybridization of DjAtg8 paralogs showed that all three genes are expressed in intestine, suggesting their conserved function in endocytosis and lysosomes. In addition, γ‐irradiation induced the up‐regulation of DjAtg8s expression and the lipidation of DjAtg8A, indicates the autophagy pathway is conserved and activated in planarian under stress. Our preliminary data showed different phenotypes in planarians with depletion of various DjAtg8 genes, and provided evidence for the putative function of Atg8 proteins in planarian digestion system and response to stress, instead of regeneration.
The Golgi Arf‐GEFs Gea1 and Gea2 are regulated by their HDS3 domains and the Rab GTPase Ypt1 (LB157)
Authors: Gustafson Margaret , Fromme J. Chris
Published: June 2021

The Golgi Arf GTPases control the sorting of membranes and proteins through all eukaryotic cells, recruiting adapters and cargo to sites of vesicle formation when in their activated GTP‐bound form. Arf guanine nucleotide exchange factors (Arf‐GEFs) are the master regulators of Arf activation and are conserved throughout eukaryotes. Two Arf‐GEFs, Gea1 and Gea2, function at the cis‐Golgi in Saccharomyces cerevisiae, but we know relatively little about how these GEFs are regulated. Recent work with their close relative at the trans‐Golgi network, Sec7, has revealed that recruitment of Sec7 to the trans‐Golgi network and relief of its autoinhibition are dependent on its C‐terminal domains, as well as on positive feedback from Arf1 and other protein interactions. The Golgi Arf‐GEFs share predicted domain architecture, suggesting that Gea1/2 may be regulated by mechanisms similar to those of Sec7. However, the specific proteins which orchestrate the activation and localization of Sec7 do not appear to act upon Gea1/2. In vitro biochemical studies, genetics, and live cell microscopy have revealed two contributing factors in Gea1/2 regulation: an interaction with the Rab GTPase Ypt1 and the regulatory roles of one of the domains of Gea1/2. Combined with structural studies, these results will define general features of Golgi Arf‐GEF regulation and localization, as well as features specific to Gea1 and Gea2, providing fundamental insight into how Arf1 is regulated at the Golgi.

Grant Funding Source: Supported by NIH/NIGMS grant R01GM098621 and NIH grant T32GM007273
Dynamic interactions between the G protein, Gαs, lipid rafts and adenylyl cyclase are altered by chronic antidepressants, cholesterol removal, cytoskeletal modification and acylation or prenylation (LB159)
Authors: Rasenick Mark , Schappi Jeffrey , Czsyz Andrew
Published: June 2021

Numerous studies suggest that chronic, but not acute antidepressant treatment increases physical coupling between Gαs and adenylyl cyclase resulting in increased cAMP signaling. Biochemical studies have demonstrated that this is due, at least in part, to the translocation of Gαs from lipid rafts into non‐raft membrane fractions where it is more effectively coupled to adenylyl cyclase. Although this effect is highly reproducible, 4° C lipid raft extractions require millions of cells and are criticized because they may not reflect the same physiology as living cells (whether or not detergents are used to extract lipid raft fractions). Here we describe a novel monomeric variant of our previously developed GFP‐Gαs fusion protein. The monomeric variant has allowed stable transfection of Gαs‐GFP into C6 glioma cells from which clones have been selected. Gαs‐GFP not only relocalizes out of lipid rafts similar to endogenous Gαs in response to antidepressant treatment, but can be used to study individual cells under physiologic conditions. Using Fluorescent Recovery After Photobleaching (FRAP), we observed that treatment with antidepressants, which decrease GFP‐Gαs raft association, but increase Gαs association with adenylyl cyclase, also increase recovery time. This appears due to the very slow recovery of adenylyl cyclase. Mutations to Gαs that render it cytosolic (palmitoylation deficient) shorten recovery time and mutations that augment membrane association retard recovery further. Disruption of lipid rafts or microtubules have a similar effect, but actin disruption does not alter Gαs FRAP. It is suggested that both rafts and adenylyl cyclase anchor Gαs, and removal from the former facilitates the latter.

Grant Funding Source: VA BX001149 and NIH MH067631
ECSCR enhances KDR activation and promotes proteolysis of internalized KDR (LB160)
Authors: Wilkinson George , Kilari Sreenivas , Remadevi Indulekha , Baofeng Zhao Baofeng , Pan Jing , Miao Robert , Ramchandran Ramani , North Paula , You Ming , Rahimi Nader
Published: June 2021

The endothelial cell‐specific chemotaxis receptor (ECSCR) is a cell‐surface protein selectively expressed by endothelial cells (ECs), with roles in EC migration, apoptosis and proliferation. Our previous study showed that loss of ECSCR in primary ECs reduced tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor 2/kinase insert domain receptor (KDR) but not VEGF receptor 1/FLT1. Here, we show that ECSCR biochemically associates with KDR but not FLT1 and that the predicted ECSCR cytoplasmic and transmembrane regions can each confer association with KDR. Stimulation with VEGF165 rapidly and transiently increases ECSCR‐KDR complex formation, a process blocked by the KDR tyrosine kinase inhibitor compound SU5416 or inhibitors of endosomal acidification. Triple labeling experiments show VEGF‐stimulated KDR+/ECSCR+ intracellular co‐localization in a perinuclear intracellular locale. Silencing of ECSCR disrupts VEGF‐induced KDR activation and AKT and ERK phosphorylation and impairs VEGF‐stimulated KDR degradation. In zebrafish, ecscr interacts with kdrl during intersomitic vessel sprouting.

Grant Funding Source: Supported by Children's Research Institute startup funds and American Heart Association Scientist Development Grant 11SDG5310020 (to G. A. W.).
The cholesterol content in erythrocytes expressing Miltenberger antigen subtype III (Mi.III) has limited effects on membrane deformability (LB161)
Authors: Hsu Kate , Yao Chin‐Che
Published: June 2021

The Miltenberger antigen subtype III (Mi.III, GP.Mur), is one of the most important red blood cell (RBC) antigens in Southeast Asia. GP.Mur is a glycophorin B‐A‐B fusion protein evolved from homologous recombination of glycophorin A and glycophorin B. We previously showed that GP.Mur in Mi.III+ cells enhances the expression of band 3, consequently facilitating Cl‐/HCO3‐ transport, CO2 respiration, and membrane resistance to osmotic stress. The aim of this study was to further investigate if the unique expressions of GP.Mur and band 3 in Mi.III also influenced cell membrane deformability. The methods utilized were osmotic gradient ektacytometry, osmotic fragility tests and Amplex Red Cholesterol assay. A total of 114 fresh RBC samples, including 50 Mi.III+ and 64 non‐Mi.III (control), were evaluated. From osmolarity fragility tests, Mi.III+ RBCs consistently exhibited superior resistance to osmotic stress. Mi.III+ RBCs however were less deformable than the control cells. The discordance between the results of membrane deformability and membrane's resistance to osmotic stress suggests that the higher band 3 expression on the Mi.III membrane likely affect the physical properties of the membrane through higher densities of band 3 interactions with adjacent lipids and cytoskeletal proteins. We also only observed the rigidifying effects of cholesterol on membrane deformability in the control cells, and not in Mi.III. We thus conclude that in contrast to the non‐Miltenberger RBCs, the cholesterol content of Mi.III+ cells has relatively limited effects on membrane deformability, presumably due to the higher band 3 levels on the Mi.III membrane.

Grant Funding Source: Supported by Taiwan National Health Research Institute, NHRI‐EX102‐10122SI
Overexpression of the oncoprotein BCL‐XL inhibits thioridazine‐induced apoptosis in acute promyelocytic leukemia cells (LB162)
Authors: Moraes Vivian , Rodrigues Tiago
Published: June 2021

The development of resistance to chemotherapeutic agents is one of the main problems in Oncology. Therefore, the improvement of novel antitumor drugs and the knowledge of new drugs and/or new targets in tumor cells are the major goal in this field. During the last years, mitochondria have emerged as an important cancer drug target, since they control several processes that are disrupted in tumor cells, including the control of cell death/survival by the BCL‐2 family proteins. Studies from our group have evidenced relevant biological effects of the antipsychotic thioridazine (TR) in tumor cells associated to mitochondrial dysfunction. In this work we evaluated the effects of BCL‐XL overexpression in the TR‐induced cell death in an acute promyelocytic leukemia model. Parental and overexpressing BCL‐XL HL‐60 cells were cultivated in RPMI1640 plus 10% BFS and antibiotics. Cell viability was evaluated by MTT and trypan blue assays. Also, cells were double stained with Annexin V‐FITC/PI and analyzed by flow cytometry. ROS generation and mitochondrial membrane potential were estimated by flow cytometry using CM‐H2DCFDA and JC‐1, respectively. The incubation of HL‐60 cells with TR for 24h resulted in cell death with an EC50 of 1.9 µM. The double staining cytometry revealed a predominant annexin V‐FITC positive and PI negative staining, indicative of apoptosis. TR‐induced HL‐60 cell death was accompanied by ROS generation and dissipation of mitochondrial transmembrane potential. The overexpression of BCL‐XL increased 7‐fold the EC50 (14.8 µM) for TR‐induced cell death and abolished ROS generation and mitochondrial depolarization. The induction of apoptosis in this model highlights the antitumor potential of TR and these results demonstrate the involvement of mitochondria and ROS unbalance in the TR‐induced apoptosis, as well as its regulation by BCL‐XL.

Grant Funding Source: Supported by FAPESP
Estrogen Increases the Mitochondria Inner Membrane Protein (Mitofilin) level after Ischemia/Reperfusion (LB163)
Authors: Harris Tanoya , Olde Bjorn , Leeb‐Lundberg Fredrik , Toro Ligia , Stefani Enrico , Bopassa Jean‐Chrisostome
Published: June 2021

Introduction: Estrogen effect can be mediated by three receptors: Classical estrogen receptors: alpha (ERα) and beta (ERβ), and recently identified G protein‐coupled estrogen receptor1 (GPER1).

Hypothesis: We investigated the role of ERα, ERβ and GPER1 in mediating rapid estrogen‐induced cardioprotection in male mice hearts subjected to ischemia/reperfusion using wild type (WT) and gene specific knockout animals.

Methods: Isolated hearts from wild type (WT: C57BL/6NCrL), ERα‐/‐, ERβ‐/‐ and GPER1‐/‐ were perfused using Langendorff apparatus with Krebs Henseleit buffer (control) or with the addition of estrogen (40 nM). Hearts were subjected to 18 min global ischemia followed by 60 min reperfusion. Cardiac function was recorded during the entire experiment and myocardial infarct size measured by TTC staining at the end of the reperfusion. Mitochondria calcium retention capacity (CRC) required to induce the mitochondrial permeability transition pore (mPTP) opening was assessed after 10 min reperfusion. Protein levels were measured by Western Blot in whole heart lysates after 5 min treatment just before ischemia, and after 10 min reperfusion. LY294002, U0126 and Chelerythrin‐Cl were used as inhibitor of PI‐3K/Akt, MAPK/ERK and PKC translocation, respectively.

Results: In WT, ERα‐/‐ and ERβ‐/‐, estrogen treatment significantly improved cardiac functional recovery, reduced infarct size and improved mitochondrial CRC. However, estrogen effects were completely absent in GPER1‐/‐. Estrogen treatment during 5 min before ischemia induced up‐regulation of Akt, GSK‐3β, and ERK1/2 phosphorylation in WT mouse as compared with control but not in GPER1‐/‐. However, after 10 min reperfusion estrogen effect was still oserved on GSK‐3β, but not on Akt and ERK1/2,. Chelerythrin‐Cl prevented estrogen‐induced cardioprotection effect and U126 abolished estrogen effect on mitochondrial CRC while LY294002 could not prevent estrogen effect on GSK‐3β observed in WT. P<0.05 and n=3‐6.

Conclusion: Rapid activation of GPER1 induces cardioprotection effect against ischemia/reperfusion injury. Estrogen effects through GPER1 are associated with phosphorylation of Akt, GSK‐3β and ERK1/2, translocation of PKC, and inhibition of the mPTP opening.

Grant Funding Source: American Heart Association
Proteomic profiling identified multiple short‐lived members of the central proteome as the direct targets of the addicted oncogenes in cancer cells (LB179)
Authors: Qi Tonggang , Zhang Wei , Luan Yun , Kong Feng , Xu Dawei , Cheng Guanghui , Wang Yunshan
Published: June 2021

Oncogene addiction is an unexplained phenomenon in the area of cancer targeted therapy. In this study, we have tested a hypothesis that rapid apoptotic response of cancer cells following acute inhibition of the addicted oncogenes is due to loss of multiple short‐lived proteins whose activity normally maintain cell survival by blocking caspase activation directly or indirectly. It was shown that rapid apoptotic response or acute apoptosis could be induced in both A431 and MiaPaCa‐2 cells, and quick down‐regulation of 17 proteins, which were all members of the central proteome of human cells, was found to be associated with the onset of acute apoptosis. Knockdown of PSMD11 could partially promote the occurrence of acute apoptosis in both MiaPaCa‐2 and PANC‐1 pancreatic cancer cells. These findings indicate that maintaining the stability of central proteome may be a primary mechanism for addicted oncogenes to maintain the survival of cancer cells through various signaling pathways, and quick loss of some of the short‐lived members of the central proteome may be the direct reason for the rapid apoptotic response or acute apoptosis following acute inhibition of the addicted oncogenes in cancer cells. These findings we have presented can help us better understand the phenomenon of oncogene‐addiction and may have important implications for the targeted therapy of cancer.

Grant Funding Source: Supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry
Ubiquitination of the transcription factor c‐maf is mediated by multiple lysine residues (LB188)
Authors: Zhang Zubin , Chen Guodong , Xu Xin , Tong Jiefei , Han Kunkun , Cao Biyin , Moran Michael , Mao Xinliang
Published: June 2021

The transcription factor c‐MAF belongs to the family of basic leucine zipper transcription factors including c‐fos and c‐jun. Previous studies suggested that c‐Maf could be polyubiquitinated followed by degradation in the proteasomes, but the mechanism is not defined. Theoretically, any lysine residues in c‐MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residues mediating c‐MAF ubiquitination. Through a series of mutational screenings from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c‐MAF ubiquitination, and a single lysine residue failed to mediate c‐MAF ubiquitination, which indicated that multiple lysine residues were required for c‐MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c‐MAF ubiquitination, which was confirmed at the cell‐based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter‐driven luciferase activity, but they were less potent than the lysine‐free counterpart (M14). Therefore, we demonstrated that c‐MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 are sufficient but not the only duo in mediating the c‐MAF ubiquitination. Moreover, c‐MAF was found to be degraded by lysosomes. This study added a novel insight for c‐MAF functional modularity, suggesting that c‐MAF stability is strictly regulated.

Grant Funding Source: National Natural Science Foundation of China
Ubiquitin‐conjugating enzyme UbcD4 as an essential component of Drosophila immune deficiency pathway (LB192)
Authors: Park Eun Sil , Kim Young‐Joon , Yoo Yung Joon
Published: June 2021

Ubiquitination has been emerged as a key regulatory mechanism in the NF‐κB pathway, in which the different types of ubiquitin chains are involved in the discrete steps. Given that the ubiquitin conjugating enzymes (E2s) are key enzymes to determine the chain topologies during ubiquitination, we tried to systematically screen out the E2s required for the IMD pathway in Drosophila, which is very similar to TNF‐α mediated NF‐κB pathway in mammals, via RNAi‐mediated knockdown of individual E2 using GAL4/UAS system. For this purpose, we first generated a transgenic fly containing a reporter dipt‐GPF gene and a fat body driver hml‐Gal4 gene that expressed the reporter protein GFP upon challenge of Gram‐negative bacteria, Ecc15. When dipt‐GFP, hml‐Gal4 transgenic flies were crossed to each of 35 UAS‐E2‐RNAi IR lines obtained from VDRC and the offspring female adult flies were infected with Ecc15, and the expressed GFP in each IR offspring was compared by Western immunoblot, it was found that the GFP level was significantly reduced in IR lines targeting UbcD4 in addition to effete and bendless. RNAi‐mediated down‐regulation of a mammalian UbcD4 homolog, Ube2K/E2‐25K, significantly attenuated the TNF‐α mediated NF‐κB activation pathway. Taken together, UbcD4 and Ube2K/E2‐25K play a conserved role as a positive regulator in Drosophila IMD pathway and mammalian TNF‐α mediated NF‐κB pathway.
3D Culture of rat embryonic stem cells and rat induced pluripotent stem cells in a novel PGmatrix hydrogel (LB20)
Authors: Rajanahalli Pavan , Huang Hongzhou , Sun Xiuzhi , Weiss Mark
Published: June 2021

Expanding embryonic stem cells (ESCs) in a synthetic 3D microenvironment in vitro while maintaining the undifferentiated state has been challenging. It would be advantageous to identify a substrate which could be modified such as by changing ligands that interact with cell surface receptors or by changing the substrate’s stiffness. We developed a synthetic PGmatrix hydrogel to include these design specifications and determined whether it could support the expansion of undifferentiated ESCs in a 3D microenvironment. Rat ESCs (rESCs) and rat induced pluripotent stem cells (riPSCs) were used for evaluation. The cells were grown both in 2D and 3D in PGmatrix hydrogel containing 2 inhibitors. Dynamic rheological tests of PGmatrix hydrogel in 2i medium showed a rapid increase of storage moduli (G’ = 600 Pa for 3mM in 1 hr) indicating gelation after mixing. The mechanical strength of hydrogel was full recovered after shear‐thinning into liquid phase.

Also, the hydrogel reached a self‐supporting strength within 1000sec which is a reasonable rate to ensure cells were suspended in the hydrogel matrix before they reached the bottom of the plate. Both cell lines were cultured for more than 30 passages using regular trypsinization method to produce single cells at passage prior to plating. We harvested cells from the hydrogel for passaging by shearing the gel to liberate the cells. Phase contrast images of cells grown in 3D showed similar morphology which had rounded phase‐bright borders compared to cells grown on murine embryonic fibroblast (MEF) feeder layer. They retained pluripotent stem cell properties and showed positive staining for alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. Karyotypes of pluripotent stem cells cultured in 3D were normal after 20 passages. Embryoid bodies (EBs) could be formed by hanging drop after the cells were trypsinized from 3D cultures. We plan to compare the gene expression profiles of 2D and 3D cells and determine if the 3D cultured cells can undergo differentiation into all the 3 germ layers and form teratomas. Growing ESCs in a fully synthetic 3D culture can significant advantages for directing differentiation and examining the interactions with surface receptors. Such knowledge will enable 3D culture for their use in drug development or for encapsulation of cells for clinical use.
Down‐regulation of miR‐96 by bone morphogenetic protein signaling is critical for vascular smooth muscle cell phenotype modulation (LB202)
Authors: Kim Kwangho , Lee Seung Ho , Kang Hara
Published: June 2021

The bone morphogenetic protein (BMP) signaling pathway is critical for the induction and maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). Inactivation of BMP signaling is common in abnormalities in vascular development and in vascular proliferative conditions, such as pulmonary artery hypertension. Herein, we identify microRNA‐96 (miR‐96) as a modulator of the VSMC phenotype in response to BMP4 signaling. We show that miR‐96 is down‐regulated by BMP4 treatment, which results in the derepression of a novel target, Tribbles‐like protein 3 (Trb3). miR‐96 targets a partially complementary sequence localized in the 3’ UTR of Trb3. Trb3 is an essential positive regulator of the BMP signaling pathway and promotes contractile phenotype in VSMCs. In conclusion, our study demonstrates a novel mechanism of regulation of SMC‐specific gene expression and induction of a VSMC contractile phenotype by the BMP4 signaling pathway via suppression of the miR‐96‐Trb3 axis.
Teneurin‐4 is a novel suppressor of myogenesis (LB235)
Authors: Ishii Kana , Suzuki Nobuharu , Mabuchi Yo , Akazawa Chihiro
Published: June 2021

Teneurin‐4 is a novel suppressor of myogenesis K.Ishii, N.Suzuki, Y.Mabuchi, C.Akazawa : Depart. of Biochem. and Biophys., Grad. Sch. of Health Care Sci., TMDU, Tokyo, Japan Muscle satellite cells are maintained in the undifferentiated quiescent state, and initiate to proliferate and differentiate at the activated stage during muscle regeneration. It has been recently reported that the transmembrane protein teneurin‐4 (Ten‐4) is highly expressed in the quiescent satellite cells. However, its cellular and molecular function in myogenesis remains unknown. We therefore aimed to elucidate the function of Ten‐4 using the mouse myoblast cell line C2C12 and the primary culture of satellite cells. During differentiation of C2C12 cells, the peak of Ten‐4 expression was observed on day 2, while expression levels of myogenic differentiation markers, myogenin and myosin heavy chain (MHC), were elevated and peaked after day 5. Knocking down the Ten‐4 expression in C2C12 cells increased the expression levels of myogenin and MHC, and promoted the myotube formation 4 days after induction of differentiation. Further, Ki67‐positive proliferating cells were increased on day 2 in the Ten‐4‐knockdown C2C12 cells. Conversely, overexpression of Ten‐4 reduced the expression levels of myogenin and MHC, the formation of myotubes, and the number of Ki67‐positive cells. Together, our findings showed that Ten‐4 is a novel suppressor of proliferation and differentiation during myogenesis of C2C12 cells, which is consistent with the previous report regarding the Ten‐4 expression in quiescent satellite cells. These evidences suggest that Ten‐4 may be a crucial regulator of maintaining the stemness of muscle satellite cells.
Uncovering novel roles of tandem BRCA1 C‐terminal (BRCT) domains in DNA repair, genome stability and breast cancer (LB236)
Authors: Davis Jamaine
Published: June 2021

Pax transactivation domain‐interacting protein (PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP consists of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains required for assembly into multiple protein complexes. Mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines. Surprisingly, we’ve discovered a novel truncated isoform of PTIP, containing the last four C‐terminal BRCT domains, (PTIP‐delta). RNAi gene depletion studies reveal a post‐transcriptional origin of PTIP‐delta. Kinetic profiling of PTIP synthesis indicates that a significant portion of PTIP is processed to PTIP‐delta within hours of translation. Recombinant forms of this PTIP‐delta retain the ability to form nuclear repair foci. Biophysical characterization uncovers new oligomerization interfaces of higher order structures composed exclusively of PTIP‐delta. These studies indicate that PTIP‐delta participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair. These studies implicate new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.

Grant Funding Source: NCI K 22
Significance of α‐subunit VISITDG sequence residues in the catalytic sites of Escherichia coli ATP synthase (LB247)
Authors: Ahmad Zulfiqar
Published: June 2021

Background: F1Fo ATP synthase is the fundamental means of cellular energy production in animals, plants, and almost all microorganisms. ATP is generated by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. A typical 70 kg human with a relatively sedentary lifestyle will generate around 2.0 million kg of ATP from ADP and Pi (inorganic phosphate) in a 75‐year lifespan. ATP synthase is the smallest known biological nanomotor, found from bacteria to man. Being the smallest biological nanomotor, ATP synthase is in the forefront of nanomedicine. Nanomedicine results from nanotechnology where molecular scale nanomotors can be used to treat disease conditions. The question of paramount importance is how to build nanomachines that are compatible with living systems and can safely operate inside the body. Here we propose that ATP synthase is a workable base model for the development of nanomotors in nanomedicine usage. In order to use it as base model we must elucidate its catalytic properties. Phosphate (Pi) binding is a primary step in ATP synthesis, so understanding the molecular basis of Pi binding is an important goal. Methods: Mutagenic analysis of the catalytic site highly conserved α‐subunit VISIT‐DG sequence residues were performed to generates single and double mutants. Growth patterns were observed on limiting glucose and succinate plates. MgPi protection assay against NBD‐Cl induced inhibition was performed to reveal the direct or indirect role of residues in Pi binding. Results: Lately, we found that αSer‐347 of the VISIT‐DG sequence is the fifth important residue involved in phosphate binding as are the four catalytic site residues βLys‐155, βArg‐182, αArg‐376, and βArg‐246. Currently we are exploring the role of other VISIT‐DG sequence residues particularly αThr‐349 and αAsp‐350 in Pi binding.

Grant Funding Source: supported by NIH/GM085771