bluuebunny/crossref_metadata_2025_split huggingface dataset:
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).
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