bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystallization and preliminary X‐ray analysis of the secreted VirA effector protein from Shigella
Authors: Davis Jamaine S , Zhang Di , Cherry Scott , Tropea Joseph , Waugh David , Wlodawer Alexander 
Published: June 2021

VirA is an effector protein secreted by the type III secretion system of Shigella. VirA functions as a cysteine protease, with its catalytic cysteine residue identified by mutation to alanine. In the study of Yoshida et al. (Science. 2006; 314 (5801):985), the ability of the enzyme to degrade microtubules was tested. These authors illustrated that VirA plays a role in the intra‐ and intercellular spreading of the bacterium by selectively degrading α‐tubulin. How VirA recognizes and degrades α‐tubulin over β‐tubulin substrates remains unclear.Bacterial invasion is an essential step in the pathogenesis of shigellosis. It is suggested that VirA is essential in creating a path to enable the bacteria to move through the dense, organized cytoplasmic network of the host cell. Shigella mutants that lack virA are unable to move through the cytoplasm and the invasiveness of these virA mutants was attenuated, suggesting that VirA is essential for Shigella virulence.We report here progress on determination of the crystal structure of VirA. The enzyme was expressed in E. coli, purified to homogeneity, and crystallized using vapor‐diffusion technique. The crystals belong to the monoclinic space group C2, with unit‐cell parameters a=150Å, b=171Å, c=46Å,β =105°, and diffract to 3.0 Å resolution. With the presence of two molecules in the asymmetric unit, the Matthews coefficient (Vm) is approximately 3.2, and corresponds to a solvent content of about 61%. Determination of the crystal structure using SAD data collected from a selenomethionine derivative is in progress.
UDP‐galactopyranose mutase: an unexpected ligand‐binding mode leads to model of substrate binding
Authors: Gruber Todd D , Borrok M Jack , Westler William M , Forest Katrina T , Kiessling Laura L 
Published: June 2021

Galactofuranose residues are key components of the cell walls of numerous pathogenic bacteria. UDP‐galactofuranose (UDP‐Galf), a biosynthetic precursor of Galf containing glycoconjugates, is produced from UDP‐galactopyranose (UDP‐Galp) by the flavoenzyme UDP‐galactopyranose mutase (UGM). We have accumulated evidence that the enzymatic reaction proceeds through an iminium intermediate formed between flavin and the substrate. To understand this unique mode of catalysis, we determined the ligand‐bound structure of UGM. We report the structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP‐glucose (UDP‐Glc) at 2.45 Å resolution, in which the crystallographic asymmetric unit contains the UGM dimer in distinct closed and open modes. In the closed monomer, a short loop restructures into an α‐helix, orienting a conserved arginine residue toward the phosphoryl groups of the nucleotide sugar, and positioning the Glc moiety near the flavin cofactor. The uracil moiety stacks with a conserved tyrosine residue, rather than the previously proposed Trp160. Despite its similarity to the natural substrate, UDP‐Glc does not participate in iminium formation upon binding to the enzyme. This observation can be rationalized by the structure; the glucose ring of the nucleotide sugar appears to be oriented improperly for catalysis. From the structure, we propose a model for the UGM‐substrate complex.Supported by NIH AI063596
The Effect of SOD‐1 Deficiency and Manganese Supplementation on Oxidative Stress Resistance in Caenorhabditis elegans
Authors: Sprowl Stephanie Ann , Zou Yani , Kaplan Rachel Deanna , Huang Ting‐Ting , Srinivasan Chandra 
Published: June 2021

Oxygen free radicals (ROS) are by‐products of normal metabolism in oxygen utilizing organisms. ROS can cause damage to living cells if not removed quickly. Superoxide dismutases (SODs) are antioxidant enzymes that form the first line of defense against ROS. Not much is known about the five forms of SOD in the roundworm Caenorhabditis elegans (C. elegans). The goals of this study are to determine which SODs are essential for oxidative stress resistance and to know if manganese (Mn) supplementation could enhance resistance against oxidative stress. Wild type and individual SOD mutant worms were cultured in growth medium containing various concentrations of paraquat (a ROS generator), and survival was scored twenty hours later. Our results show that only sod‐1 (encodes SOD‐1, a CuZn‐containing SOD) mutant worms are significantly more sensitive to paraquat than the wild type controls. This is in line with our finding that SOD‐1 is the major SOD in C. elegans. Since we have shown Mn to have an antioxidant‐like property, we examined the effect of Mn on paraquat toxicity. Mn supplementation (1 mM) in the culture medium increases resistance, as measured by LD50 by at least 3‐fold in sod‐1 mutant worms. Increased paraquat resistance is also observed in wild type worms treated with Mn; however, the extent of increase is only around 2‐fold. These results lend further support to the antioxidant potential of Mn.Funding provided by NIH.
Radiation Effects on Levels of Total Mitochondrial DNA and DNA Bearing the Common Deletion in a Pearson's Syndrome Pedigree
Authors: Vrooman Scott , Pogozelski Wendy 
Published: June 2021

Mutations in mitochondrial DNA (mtDNA) correlate with aging and oxidative stress and they appear in a host of degenerative diseases. Little is known about how ionizing radiation might contribute to the appearance of these mutations, however. Here we quantify a specific deletion of 4977 base pairs (known as the “common deletion”) as a function of X‐ray dose using real‐time PCR. We grew lymphoblast cells derived from a patient with Pearson's Marrow/Pancreas syndrome (known to contain elevated levels of the common deletion) as well as normal cells derived from the patient's mother and sister. We irradiated the cells with doses of 0.5–5 Gy of gamma radiation. Total (nuclear and mitochondrial) and mitochondrial DNA extracts were then analyzed along with cloned plasmids that served as quantification controls. The deletion was only measurable in the Pearson's Syndrome cells and results showed elevated levels with radiation dose. The total mtDNA remained relatively constant in these cells. In contrast, the levels of total mtDNA increased in the normal control cells derived from the patient's mother and sister.This research is supported by a Henry Dreyfus Teacher‐Scholar Award.
Systems biology of the mammalian Unfolded Protein Response
Authors: van Riel Natal A , Tiemann Christian A , Jeneson Jeroen A , Hilbers Peter A 
Published: June 2021

The Unfolded Protein Response (UPR) is a coordinated endoplasmic reticulum (ER) stress response program. UPR is initiated whenever protein folding in the ER is reduced and accumulation of malfolded proteins takes place. ER stress is associated with diseases, including type 2 diabetes. However, the dynamics and regulation of the underlying signal transduction network are largely unknown.A first mathematical model for the UPR in mammalian cells was proposed including the PERK and ATF6 pathways. The differential equation model was parameterized from Western blot time course data of phosphorylated PERK and cleaved ATF6 after exposing cells to dithiothreitol (DTT), which prevents the formation of disulfide bonds (DuRose et al. Mol. Biol. Cell, 17: 3095–3107, 2006).Sensitivity analysis showed that only a few kinetic parameters were critical for the predicted p‐PERK and ATF6 time responses. Interestingly, variations in these parameters could move the UPR system towards different new steady‐states. In certain cases the system returned to its original steady‐state while the cells were still exposed to DTT (so‐called perfect adaptation). In other cases, the stress sensors remained active. However, the difference in endpoints only emerged after 5 hours, when no experimental data was available. This model‐based hypothesis is open for experimental testing.
Analysis of Trypanosoma cruzi Phosphoproteome by Mass Spectrometry
Authors: Gaynor Matthew R , Nakayasu Ernesto S , Almeida Igor C 
Published: June 2021

Trypanosoma cruzi is the etiologic agent of Chagas’ disease, which affects over 11 million people in Latin America. Lately, much research has been focused on drug and vaccine development against this parasite. In eukaryotic cells, protein phosphorylation regulates many vital pathways and has been extensively studied as potential molecular target. Here, we analyzed T. cruzi phosphoproteins and mapped the phosphorylation sites through mass spectrometry (MS). Total proteins from T. cruzi epimastigote forms were digested with trypsin/urea and fractionated in a strong cation‐exchange ziptip. Ensuing phosphopeptides were purified by immobilized metal‐affinity chromatography (IMAC) and analyzed by liquid chromatography‐tandem MS (LC‐MS/MS, ‐MS3, ‐pseudoMS3). The resulting spectra were searched against T. cruzi sequences in the GenBank using Sequest algorithm. We were able to identify 143 phosphorylation sites from 98 distinct proteins. The Gene Ontology analysis revealed several proteins with protein‐ and nucleotide‐binding activity, cytoskeleton proteins, and proteins involved signal transduction. To our best knowledge, this is the first phosphoproteome study performed in a pathogenic protozoan. Our data may provide new insights into discovery of new interventions to prevent or treat Chagas’ disease.Supported by NSF/REU Program (grant#DBI‐0353887), and NIH/SCORE (grant#2SO6GM0812‐37).
Modeling Tumor Development: the Role of L1
Authors: Brady Owen Adam , Yang Muhua , Galileo Deni S. 
Published: June 2021

L1 is a membrane‐bound neural cell adhesion protein involved in the development of the nervous system. However, L1 has been implicated in mediating some of the invasive properties of several cancers including gliomas and breast cancers when proteolyzed to create a soluble ectodomain, which then binds to cell surface integrin receptors in an autocrine/paracrine manner. L1 has also been shown to rescue cancerous cells from apoptosis. It was hypothesized that polyclonal antibodies raised against a 15 amino acid peptide corresponding to a critical integrin binding region of human L1 would slow the migration of rat 9L/LacZ and human T98 G glioma cells in vitro, whereas the peptide alone would stimulate migration. Incubation of cells in the peptide decreased cellular migration, possibly due to competitive inhibition. The effects of the antibody on cell migration were inconclusive. Quail QT6 cells have been stably transfected with an L1 ectodomain‐Fc fusion protein (L1‐Fc) designed to be secreted into the media. Once purified, it will be applied to 9L/LacZ and T98 G cells and analyzed using automated time‐lapse microscopy. Additionally, glioma cell migration will be analyzed while on top of QT6 cells secreting L1‐Fc. Both conditions are hypothesized to increase the migration rates of these cell lines by mimicking the activity of endogenous L1 ectodomains.Supported by HHMI and INBRE.
Ski is required for normal development of embryonic progenitors of hypaxial muscle
Authors: Zhang Hong , Chen Yishi , Colmenares Clemencia , Stavnezer Edward 
Published: June 2021

The Ski encodes a transcriptional co‐regulator that stimulates muscle differentiation. We have found that Ski−/− embryos exhibit selective defect in hypaxial muscles. Hypaxial muscles are derived from progenitor cells that originate in the dermomyotome, migrate to distal sites and commit to a myogenic fate. However, the Ski−/− embryos showed that the dermomyotome developed normally. We found that myogenic determination and terminal differentiation were initiated appropriately in the absence of Ski. However, the number of cells expressing myogenic regulatory factors (MRF) genes was reduced and they were restricted to proximal limbs. Our study shows that this deficiency in number and distribution of MRF+ cells is not caused by abnormal proliferation, apoptosis or differentiation. Further examination of Pax3 and Met, which are important for progenitor cell migration, indicated that this defect resulted from impaired migration of myogenic progenitors.This work was supported by NIH grants (DE15198 to C.C and E.S, CA43600 to E.S, and HD30728 to C.C).
Variants of 5‐lipoxygenase from human neutrophils impact on leukotriene biosynthesis in HEK293 cells
Authors: Boudreau Luc H , Bertin Jonathan O , Surette Marc E 
Published: June 2021

5‐lipoxygenase (5‐LO) catalyzes the oxygenation of arachidonic acid and is an essential enzyme forleukotriene (LT) biosynthesis. Human neutrophils (PMN) are important producers of LTB4, a potent chemoattractant involved in the inflammatory response.In this study, we evaluated whether alternative forms of 5‐LO mRNA exist in human PMN and if they are implicated in the biosynthesis of LTB4. RT−PCR analysis of total mRNA from human PMN revealed several potential variants of 5‐LO mRNA. Molecular cloning and sequencing experiments showed that at least three isoforms are present in human PMN. One containing all 14 exons with the expected splicing sites (full 5‐LO) producing a known protein of 78 kDa. Another isoform retained intron 10 (alpha‐10) resulting in a shift of the open reading frame and produces a protein of expected molecular weight of 56 kDa. A third isoform lacked the first 96 nucleotides of exon 10 (delta‐p10) and produces a protein of expected molecular weight of 74 kDa. HEK293 cells transfected with the full 5‐LO produced LTB4 and 5‐hydroxyeicosatetraenoic acid (5‐HETE) following ionophore stimulation; however, cells transfected with either alternative isoform produced no 5‐LO products. HEK293 cells transfected with the full 5‐LO and co‐transfected with the alpha‐10 or the delta‐p10 isoform produced 25±2% (mean±sem) and 49±7% (mean±sem) less 5‐LO products, respectively, compared to cells expressing only the full 5‐LO. These alternative isoforms of 5‐LO may represent a new mechanism for the regulation of LT biosynthesis in inflammatory cells. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program).
Modulation of Lipid Body Biogenesis in Epithelial Cells
Authors: Diaz Bruno L , Moreira Luciana S , Piva Bruno O , Bozza Patricia T , Bandeira‐Melo Christianne 
Published: June 2021

Lipid Bodies are cytoplasmic inclusions mainly formed by triglycerides and cholesterol esters, being also intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoids generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in epithelial cells. A rat cell line derived from normal intestinal epithelium, IEC‐6, was used. IEC‐6 in sub‐confluent cultures presented 15–20 lipid bodies/cell after staining with by OsO4. Lipid bodies were virtually absent in IEC‐6 upon reaching confluency or after FBS withdraw. Stimulation of confluent IEC‐6 with IL‐1β, PAF, or PMA was unable to induce lipid bodies. In contrast, AA dose‐dependently induced lipid body formation and proliferation foci in confluent cultures independently of its metabolism to PGE2. AA induction of lipid bodies was dependent on signaling through p38, PKC, and PI3K, but not ERK 1/2 or JNK. Lipid body biogenesis facilitates AA release after ionophore activation without modifying cPLA2α expression. Our results suggest a strong relationship between lipid body biogenesis and epithelial cell proliferation, and indicate that these organelles are induced in a stimulus‐specific manner and facilitate AA mobilization in epithelial cells.Financial support: CNPq, FAPERJ, CAPES
Time Course Analysis of Eight Autophosphorylation Sites on p21‐Activated Protein Kinase Pak2
Authors: Jung Jin‐Hun , White Jon , Traugh Jolinda 
Published: June 2021

The p21‐activated protein kinase Pak2 is activated under cytostatic or apoptotic conditions. Binding of Cdc42(GTP) stimulates autophosphorylation of Pak2 up to 8 mol/mol, which is required for full activation. There are seven serine sites (S19, S20, S55, S141, S165, S192 and S197) in the regulatory domain, and one threonine (T402) in the activation loop. The time course of autophosphorylation of Cdc42‐activated Pak2 showed a gradual increase from 1 to 30 min. When autophosphorylated Pak2 was subjected to two‐dimensional tryptic phosphopeptide mapping and quantified, S197 was rapidly phosphorylated during first 10 min and constituted about 40% of the autophosphorylation, while the other sites were significantly less. To delineate the effect of each site in the molecular activation of Pak2, the sites were changed to alanine by site‐directed mutagenesis and the level of autophosphorylation was analyzed. In the presence of Cdc42, the four alanine mutants, S141A, S192A, S197A and T402A, showed a 50–60% reduction in autophosphorylation, while there was no effect on S19A/S20A, S55A and S165A, as compared to the level with WT Pak2. Thus, autophosphorylation of S141, S192, S197 and T402 are required to achieve maximum autophosphorylation, and phosphorylation of S197 contributes to promoting further autophosphorylation on other sites.Supported by NIH grant GM 26738 to J. A. T.
Cdc42/Rac function in NK cells and CTLs is variable and governed by spatiotemporal patterning of Cdc42/Rac
Authors: Sinai Parisa , Nguyen Chau , Schatzle John D. , Wuelfing Christoph 
Published: June 2021

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are central immune effectors. They activate in a cellular interaction with target cells, leading to target cell lysis and IFN? secretion. These effector functions require effective cellular polarization. We have therefore studied roles of Cdc42 and Rac, two general regulators of cellular polarization, in primary NK cells and CTLs. NK cells were primed with IL‐2, IL‐12/IL‐18, or IL‐15, reflecting different physiological and therapeutically used activation conditions. We expected that Cdc42/Rac by promoting cellular polarization would support effector functions. Surprisingly however, functions of Cdc42/Rac varied dramatically between the different cell types and activation conditions. When we determined the spatiotemporal patterns of Cdc42/Rac activity in NK cell/CTL – target cell interactions, we intriguingly found that Cdc42/Rac could only support effector functions when enriched at the center of the effector/target cell interface. Our data thus establish that Cdc42/Rac function in NK cells and CTLs is not only governed by the activation of these proteins but, as importantly, by the location of this activation.Supported by the NIH
Important roles for Fyn in CD4 T cell activation and helper functions in vivo
Authors: Yusuf Isharat , McCausland Megan , Tran Hung , Crotty Shane 
Published: June 2021

Fyn is a src‐family kinase involved in signaling downstream of the T cell receptor and B cell receptor. Initial reports on the Fyn‐deficient mice revealed no obvious defects in mature CD4 T cells suggesting that its function maybe redundant with Lck. More recently Fyn has been shown to play a critical role downstream of the SLAM‐family receptors through the adaptor protein, S AP/SH2DIA. Here we report that although early CD4 T cell dependent antibody production appears to be normal, Fyn‐deficient mice display a defect in germinal center formation and long‐term antibody production resulting from a lack of long‐lived plasma cells – a defect similar to SAP‐deficient mice. These defects are distinct from Fyn's role downstream of SLAM‐SAP signaling pathway as demonstrated by the use of a SAP mutant incapable of binding Fyn. This non‐Fyn binding mutant SAP is fully capable of rescuing the humoral defect seen in the SAP‐deficient mice. The data here suggest that Fyn may indeed play a critical role in CD4 T cell help to B cells, but Fyn's role in the humoral response is independent of the SLAM‐SAP pathway.This work was supported by NIH T32 AI060536 grant (UCSD) to IY
AID‐ and Ung‐dependent generation of staggered double‐strand DNA breaks in class switch DNA recombination: a post‐cleavage role for AID
Authors: Zan Hong , Casali Paolo 
Published: June 2021

Double‐strand DNA breaks (DSBs) are critical for Ig class switch (S) DNA recombination (CSR). They would be generated through deamination of dC by AID, subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and nicking by apurinic/apyrimidic endonuclease of nearby abasic sites on opposite DNA strands. We found that in human and mouse B cells, S region DSBs can be generated in an AID‐ and Ung‐independent fashion. These DSBs were blunt and 5′‐phosphorylated, and could be readily detected in human and mouse AID‐ or Ung‐deficient B cells. In B cells undergoing CSR, blunt DSBs were processed in an AID‐ and Ung‐dependent fashion to yield staggered DNA ends. Accordingly, we readily detected blunt‐DSB in B cells from AID‐ or Ung‐deficient patients and AID‐ or Ung‐deficient mice. These B cells were CSR defective, but showed evidence of intra‐S region recombination. Enforced expression of AID in AID‐negative B cells converted S region blunt DSBs to staggered DSBs. Conversely, enforced expression of dominant negative AID or inhibition of Ung by Ung inhibitor (Ugi) in switching B cells abrogated the emergence of staggered DSBs and concomitant CSR. Thus, AID and Ung generate staggered DSBs not only by cleaving intact double‐strand DNA, but also by processing blunt DSB ends, whose generation is AID‐ and Ung‐independent, thereby outlining a post‐cleavage role for AID in CSR.Supported by NIH grants AI 45011, AI 60573 and AR 40908.
Ceramide Disrupts HLA Class II‐restricted Antigen Processing and Presentation
Authors: Haque Azizul , Hajiaghamohseni Laela M. , Harbeson Caroline , Amria Shereen , Xiang Liu , Bielawska Alicja , Hannun Yusuf , Norris James S. 
Published: June 2021

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effect of ceramide on antigen processing and HLA class II‐mediated delivery to CD4+ T cells remains unclear. We have previously shown that intracellular ceramide levels can be regulated by ceramide anlogs that directly interfere with ceramide‐metabolizing enzymes such as acid ceramidases. In this study, we examined the actions of these cell permeable ceramide analogs on professional antigen presenting cells (APC) that constitutively express HLA class II proteins and functionally present antigens to CD4+ T cells. Our results show that the treatment of APC with ceramide analogs perturbs Ag processing and presentation in the context of HLA class II molecules. Cellular uptake of ceramide analogs into the endolysosomal compartments did not alter intracellulaer class II protein levels as confirmed by confocal microscopy and western blot analysis. Mass spectroscopic analysis showed that ceramide analogs decreased the activity of acid ceramidase elevating intracellular ceramide levels in the APC. Western blot and flow cytometric analysis confirmed that the ceramide analogs diminished acid ceramidase protein expression in APC, but not cell surface class II molecules. Data also showed that the elevation of intracellular ceramide modulates endolysosomal proteases as well as the components of the HLA class II pathway. These findings suggest that ceramide regulates multiple pathways in APC including antigen processing and presentation via HLA class II molecules.Supported by the Leukemia and Lymphoma Society (3024), American Lung Association (RG‐10435‐N), MUSC HCC Funds and NC1 PO1 CA97132