bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of a Mitochondrial Twin Arginine Transport Pathway in Arabidopsis thaliana
Authors: Weerakoon Tasmeen Shiny , Ma Qianqian , Dabney‐Smith Carole 
Published: November 2023

Most proteins found in organelles such as chloroplasts or mitochondria are synthesized at a location different to their site of function. Cells therefore, must have efficient protein routing processes to allow proteins to go from their site of synthesis to their site of function, which may involve crossing multiple membrane systems. One such vital protein transport pathway is the Tat (Twin Arginine Translocation) pathway. The Tat pathway has been found in bacteria, archaea and eukaryotic organelles such as the chloroplast. The chloroplast Tat pathway (or analogous Tat pathways in bacteria) is capable of transporting fully folded proteins across membranes using only a proton motive force instead of ATP as an energy source. It is composed of three protein components, namely cpTatC, Tha4 and Hcf106 that work together to allow the transport of precursor proteins containing an obligate twin arginine motif in the targeting sequence. However, recent experiments using Nicotiana tabacum cells showed an up‐regulation of a mitochondrial gene coding for a putative mtTatC protein when the cells were subjected to salicylic acid treatment, which mimics a defense response in the plant to attack by pests. We have confirmed a similar upregulation under salicylic acid stress of the putative mitochondrial tatC gene in Arabidopsis thaliana. The presence and upregulation under stress of the mttatC gene suggests the possible presence of a mitochondrial Tat pathway. However, single nuclear genes encode the other cpTat components, which suggests either of the other components would likely dual localize to both organelles when the mtTat pathway was functional. To test for this, we have transformed Arabidopsis thaliana with Tha4‐GFP constructs and looked for its localization in the presence or absence of various biotic or abiotic stresses. Furthermore we are investigating mtTatC and Tha4 protein expression levels at different concentrations of salicylic acid in whole leaf tissue extract and in isolated mitochondria. Evaluating the presence of a mitochondrial Tat pathway will shed light on a unique protein sorting and transport mechanism in plant mitochondria, a facility previously thought to be lost during the mitochondrial evolutionary process.Support or Funding InformationDr. Dabney‐Smith Group, Center for Bioinformatics and Functional Genomics (CBFG, Center for Advanced Microscopy and Imaging (CAMI), DOE Office of Science Early Career Research Program (DE‐FG02‐10ER16138)
System xc− Regulation: Effects of Mutations on Membrane Expression and Ubiquitination Status
Authors: Schmidt Mackenzie , Gibson Amanda , Hardy Emma , Versluis Philip , Lang Sara , Georges Anne , Chase Leah 
Published: November 2023

System xc− exchanges intracellular glutamate for extracellular cystine across the membrane of many cell types, including astrocytes. Its activity directly regulates the synthesis of the antioxidant glutathione and the extracellular concentration of glutamate in some areas of the brain. Dysregulation of the transporter can lead to excessive glutamate release and excitotoxic cell death or the depletion of glutathione stores and the development of oxidative stress. We recently demonstrated that oxidants acutely upregulate System xc− activity by triggering the rapid redistribution of the transporter from intracellular compartments to the cell surface. Our current work suggests that the trafficking of the transporter may be regulated by ubiquitination and that oxidant exposure directly influences the ubiquitination of the transporter. Since increased ubiquitination tends to decrease the cell surface expression of many membrane transporters, we sought to test the hypothesis that System xc− is ubiquitinated and that the ubiquitination status of the transporter regulates both its cell surface expression and activity. We have used a mutagenesis approach to disrupt putative ubiquitination sites and a putative ubiquitin ligase binding site within a myc‐tagged System xc− construct so that we can understand the role ubiquitination plays in regulating the cell surface expression of System xc−. There are seven highly conserved lysine residues within xCT that are located on the cytoplasmic side of the membrane. These residues are located at positions 4, 37, 41, 43, 422, 472, and 473. We have created mutant forms of this construct containing single or multiple lysine to arginine mutations so that we could assess the effect of these mutations on cell surface expression of System xc‐. Using biotinylation assays and immunocytochemical analysis, we have demonstrated that mutation of the N‐terminal lysine residues increases the cell surface expression of the transporter. We are currently assessing the ubiquitination status of these mutant transporters to determine if the changes in ubiquitination of the transporter are associated with changes in the cell surface expression and activity of the transporter. In addition, we have identified a putative ubiquitin ligase binding site in the C‐terminus of the transporter. Disruption of this binding site also leads to an increase in cell surface expression of the transporter. Collectively, these data suggest that System xc− is regulated by changes in its ubiquitination status such that factors which lead to diminished ubiquitination will allow for increased cell surface expression of the transporter.Support or Funding InformationThis work was supported by RUI 0843564.
Optimization for Transfection and Isolation of C‐Mannosyltransferase DPY19‐L1
Authors: Murdock Brandon 
Published: November 2023

Glycosyltransferases are a unique class of enzymes that catalyze the attachment of sugar molecules to proteins as posttranslational modifications. The three ways in which sugar moieties are attached to proteins are by N‐linked, O‐linked, or C‐linked glycosylation. The mannosyltransferase DPY19‐L1 is a mammalian enzyme that attaches a mannose sugar to the second carbon of the indole ring of tryptophan residues in a tryptophan‐x‐x‐tryptophan consensus sequence.The DPY19‐L1 mannosyltransferase has never been isolated, which is one of our main goals. To accomplish this task, a HEK‐293 cell line was transfected with purified DPY19‐L1 plasmid utilizing cationic lipids to facilitate the uptake of the purified plasmid. Synthesized linear polyethylenimine (PEI) was the cationic lipid used due to its cost efficiency and ease of synthesis. A number of transfection conditions were screened to determine that efficiency was optimized at high confluence and a PEI to DNA ratio of 7:1. Once the protein had been produced, the enzyme was isolated utilizing a his‐tag (hexahistidine tag) on the N‐terminus of the C‐mannosyltransferase. The his‐tag facilitated binding of the protein to the nickel lining of the column during affinity chromatography. The purity of the isolated protein was then verified using SDS‐PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).The long‐term purpose of this research is to determine the enzymatic mechanism by which DPY19‐L1 catalyzes the covalent linkage of mannose to tryptophan residues. For this, reverse phase HPLC (high performance liquid chromatography) will be utilized to determine the enzymatic activity of DPY19‐L1 by measuring the rate of product formation. Subsequently, the residues in the enzyme active site will be analyzed to determine the mechanism by which tryptophan residues are mannosylated.
Cellulase‐Polymer Bioconjugates to Improve Enzyme Stability for Biofuel Production
Authors: Stewart Jamie , Wright Thai , Schmitz Benjamin , Williams Cameron , Makaroff Katherine , Konkolewicz Dominik , Page Richard C 
Published: November 2023

Protein polymer hybrids are bioconjugates that offer myriad customizations. The synthesis of these molecules allows for the opportunity to enhance proteolytic stability, manipulate the pH and temperature optimums of enzymes, and construct new and exciting biomaterials. To meet the increasing demands for biofuels, including mandates set by the US Renewable Fuel Standard II (RFS2), new enzymes with improved functionality and stability are desperately needed. Chief among these needs is the RFS2 directive that sets a target of increasing bioethanol production 600‐fold by 2022 and sets a cap on bioethanol that can be produced from grains. In contrast, cellulosic bioethanol produces glucose through enzyme‐mediated degradation of cellulose to produce a precursor that can be used for ethanol production by fermentation. Cellulosic bioethanol production therefore stands as an opportunity to capitalize on an under‐utilized resource, cellulose, to produce bioethanol. To increase the rate of cellulosic bioethanol production we have utilized the cellulase FnCel5a from thermophilic Fervidobacterium nodosum. We have produced cellulase‐polymer conjugates with improved chemical stability. Utilizing site‐directed mutagenesis we control the location of the polymer placement. Herein we present biochemical and biophysical analyses of cellulase‐polymer conjugate stability and activity conferred by interactions between the cellulase and conjugated dimethylacrylamide‐ or acrylamide‐based polymers. Our research seeks to achieve cost‐effective mass production of glucose from cellulose. As the demand for renewable energy increases, the need for rapid and efficient production of this renewable fuel source precursor is critical.Support or Funding InformationThe authors acknowledge financial support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).
Electrostatic Contributions of Aromatic Residues in Protein‐Carbohydrate Interactions
Authors: Early Stephen A , Diehl Roger C , Kiessling Laura L 
Published: November 2023

Protein‐carbohydrate interactions play critical recognition and signaling roles in a diverse array of biological processes—from stem cell differentiation to immune responses. However, the intermolecular forces that govern protein‐carbohydrate interactions have not been quantitatively elucidated. Given the dogma in the field and our data, the goal of this study is to understand the molecular basis for aromatic‐carbohydrate interactions. Previous bioinformatic analyses have shown that aromatic residues are markedly conserved and overrepresented in carbohydrate binding sites while aliphatic residues are dramatically underrepresented. Unlike aliphatic residues, aromatic amino acids present electronegative π‐electron systems that can interact with carbohydrate C–H bonds through CH–π interactions. We posited that if electrostatic contributions are important for CH–π interactions in protein–carbohydrate complexes, differences in the electronics of the aromatic systems and carbohydrate C–H bonds would determine participation in such interactions. We therefore sought to design a system to interrogate the molecular basis for aromatic‐carbohydrate interactions within proteins. Using the immunologically relevant lectin, galectin‐3, the stability and lactose binding affinity of galectin‐3 variants at CH‐π acceptor residue, W181, were assessed through differential scanning fluorimetry and isothermal titration calorimetry. We observed that the replacement of W181 with the smaller aromatic residues phenylalanine, tyrosine, and histidine diminished both protein stability and binding affinity, while replacement of W181 with an aliphatic methionine residue maintained protein stability but abrogated lactose binding. Supported by stability and binding data for the galectin‐3 variants, we conclude that CH‐π interactions are particularly important in promoting the stability of carbohydrate binding proteins as well as their affinity for binding β‐galactosides through the contribution of electrostatics versus hydrophobic interactions.Support or Funding InformationThis work was supported by a UW‐Madison L&S Honors Welton Undergraduate Research Fellowship.
Identification of a post‐translational modification with ribitol‐phosphate and its defect in muscular dystrophy: Roles of ISPD, fukutin, and FKRP in α‐dystroglycan glycosylation
Authors: Kanagawa Motoi , Kobayashi Kazuhiro , Tajiri Michiko , Manya Hiroshi , Kuga Atsushi , Yamaguchi Yoshiki , Wada Yoshinao , Endo Tamao , Toda Tatsushi 
Published: November 2023

Glycosylation is an essential post‐translational modification that underlies many biological processes and diseases. α‐dystroglycan (α‐DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α‐dystroglycanopathies). Here we identify the glycan unit ribitol 5‐phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α‐DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand‐binding moiety. We show that enzyme activities of three major α‐dystroglycanopathy‐causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain‐containing (ISPD) is cytidine diphosphate ribitol (CDP‐Rbo) synthase. Fukutin and fukutin‐related protein are sequentially acting Rbo5P transferases that use CDP‐Rbo. Consequently, Rbo5P glycosylation is defective in α‐dystroglycanopathy models. Supplementation of CDP‐Rbo to ISPD‐deficient cells restored α‐DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α‐DG‐associated diseases.Support or Funding InformationJapan Society for the Promotion of Science 15H04352
A Novel Hemolysin with Anti‐cancer and Anti‐fungal Properties Binds to Serum Glycoproteins and Cholesterol
Authors: Welch Christina , Fan Ni , Brown Robert , Talaga Melanie , Fueri Ashli , Driscoll Kyle , Lawry Kevin , Vizurraga Alexander , Rekhi Ramandeep , Bandyopadhyay Purnima , Dam Tarun 
Published: November 2023

Lysins are capable of disrupting mammalian cell membranes by specifically binding to membrane components. Lysin can be a protein, a peptide or a glycoside. Certain lysins, called hemolysins, can disintegrate mammalian erythrocytes. A few of the reported lysins can bind to specific glycan structures. Here we report, for the first time, a glycan‐binding hemolysin (named Hemolysin‐X or HelyX) from a flowering plant. HelyX was purified by ammonium sulfate precipitation, size exclusion chromatography and high performance liquid chromatography (HPLC). Activity and ligand binding properties of HelyX were studied by plate based assays and spectrophotometric analysis. Interactions of HelyX with mammalian and fungal cells were studied through microscopy and flow cytometry. HelyX showed rapid and robust hemolytic activities even at a very low concentration. The molecular weight of this hemolysin is 2 kDa. Hemolysis by HelyX was completely inhibited by serum glycoproteins (Fetuin, asialofetuin, thyroglobulin and fibrinogen) and cholesterol. Interestingly, the hemolysins of cholera bacteria were reported to show similar binding specificities. When tested with colon and breast cancer cell lines, HelyX showed significant apoptotic activities. The hemolysin also inhibited the growth of yeast cells.Support or Funding InformationMichigan Technological University
Deconvolution of Glycan Occupancy Heterogeneity for Rational HIV Immunogen Design
Authors: Yu Wen‐Han , Zhao Peng , Draghi Monia , Arevalo Claudia , Karsten Christina , Wells Lance , Lauffenburger Douglas , Alter Galit 
Published: November 2023

The HIV envelop glycoproteins (Env) that mediate viral infectivity are the major targets for broadly neutralizing antibodies (bNAbs). Mounting evidence suggests that the highly variable carbohydrate shields, accounting for more than half of Env protein mass, contribute critically to the antigenicity of HIV Env, and represent antigenic determinants for many of the most potent bNAbs. The vaccine design approaches, which are able to harness the antigenic nature of the Env glycan and to guide the vaccine‐induced humoral immune response to the key glyco‐protein epitopes, will significantly advance the field. But to date HIV immunogens continue to fail to induce antibodies with appreciable cross‐neutralizing breadth. The major obstacles are the limited understandings of proximal and distal glycans of the Env collectively impacting on bNAb antigenicity. While global HIV Env glycans are highly interconnected and coordinately shape the epitope landscape, many studies focusing on individual glycan‐bNAb interactions often overlooked the impact of the global glycans on bNAb bindings. Here we demonstrate a systems glycobiology approach to computationally deconvolute and harness the complexity of the global HIV glycan shield heterogeneity, aimed to design a novel class of immunogens with an enhanced capacity to present bNAb epitopes. Specifically, we exploited global glycosylation site occupancy profiles on 94 recombinant HIV Env glycoproteins representing a panel of global HIV isolates, linked to 16 bNAb and non‐bNAb binding profiles. Using our Bayesian machine‐learning analysis, the general roles for distal and proximal glycans in the manner of antagonizing and agonizing bNAb bindings were identified, in such a way that the bNAb‐glycan binding signature was identified for individual bNAbs. Furthermore, we developed an evolution‐based de novo immunogen design algorithm, which taking bNAb‐glycan binding signature as well as envelop sequences as prior knowledge, engineering global glycosylation of HIV envelop to achieve optimized bNAb antigenicity. A set of glycan‐optimized immunogens were designed, which selectively improved or altered specific bNAb bindings as the algorithm predicted. This approach provides a novel rational glycoengineering strategy to program HIV envelope antigenicity via alteration of global Env glycosylation to predictively modulate bNAb binding, and demonstrates a proof‐of‐concept of immunogen design for HIV and other viral glycoproteins.Support or Funding InformationThis work was supported by the Bill and Melinda Gates Foundation (OPP1097381).
Arabidopsis Plants Expressing a Fungal Pectin Methylesterase Enzyme Have Reduced Degree of Polysaccharide Methylation and Exhibit a Dwarfed Phenotype and Resistance to Stresses
Authors: Chambers Lauran , Zabotina Olga , Reem Nathan , Abdullah Siti Farah 
Published: November 2023

Pectin is a major component of the plant cell wall and is involved in functions including growth of the plant cell, organ development, and defense against pathogens. Our lab has developed a suite of transgenic Arabidopsis thaliana plants expressing microbial‐derived cell wall‐degrading enzymes to study Cell Wall Integrity (CWI) responses. Plants expressing one such enzyme, A. nidulans Pectin Methylesterase (AnPME), exhibit dwarfism beginning with emergence of the first true leaves and affecting all specialized organs and structures throughout their lifetime. These plants also possess reduced sensitivity to salt stress; while roots of AnPME plants are much smaller than wild‐type roots under unstressed conditions, root growth was not inhibited by 100 mM NaCl treatment. Preliminary results have also shown that AnPME plants are less susceptible to infection by the cyst‐forming nematode H. schaachtii. Ongoing research aims to quantify the degree of cell wall de‐methylesterification to correlate with degree of dwarfism, and further confirm resistance of roots to nematode penetration. These results illustrate the importance of pectin methylesterification status in plant fitness and development, as well as response to both biotic and abiotic stresses, most likely through CWI response.Support or Funding InformationLinder Family Undergraduate Research Fellowship, Rob Stupka Memorial Scholarship
Bifidobacterium dentium regulates intestinal mucus production and glycosylation
Authors: Engevik Melinda Anne , Luk Berkley Kristina , Visuthranukul Chonnikant , Versalovic James 
Published: November 2023

BackgroundBifidobacteria are commensal organisms in the gastrointestinal tract that are known to exhibit health‐promoting effects. In humans, bifidobacteria are detectable within the first week after birth and are a predominant genus of the infant intestinal microbiota. Previous studies have demonstrated that Bifidobacterium species are able to adhere to and colonize intestinal mucus. The intestinal mucus layer provides both a barrier and interface between the gut microbiota and the host. Mucus is secreted from goblet cells as heavily O‐glycosylated proteins. Disruption of the protective mucus layer has been implicated in several inflammatory diseases. Currently little is known regarding how specific microbes are able to modulate mucus production and glycosylation. We hypothesized that the human commensal Bifiboacterium dentium, a representative member of Bifidobacteria, would modulate O‐glycan synthesis and increase intestinal mucus production.Methods & ResultsSwiss Webster germ free mice (GF) were mono‐associated with B. dentium, while control GF mice received sterile media gavages. Standard bacterial plating, fecal gDNA and 16S fluorescent in situ hybridization (FISH) were used to confirm colonization of B. dentium. Colon tissue was collected in Trizol for RNA or fixed in Carnoy's fixative to maintain mucus architecture. Mucin mRNA was examined by qPCR and mucin proteins were examined by Periodic Acid Schiff‐Alcian Blue (PAS‐AB) and immunofluorescence. Colonoids were generated from germ free mouse colonic stem cells and grown in matrigel to generate 3‐D structure. B. dentium mono‐associated mice had a 20 fold increase in the goblet cell differentiation marker Kruppel‐Like Factor 4 (klf4) at the level of mRNA compared with GF controls. Likewise, B. dentium mice exhibited increased acidic mucin filled goblet cells as denoted by PAS‐AB stain compared with GF controls. B. dentium mice had a >5‐fold increase in the main secreted mucin Muc2 mRNA and protein, and increased the co‐secreted proteins Trefoil Factor 3 (TFF3) and resistin‐like molecule (Relmβ). Analysis of mucin O‐glycosylation genes revealed that B. dentium upregulated b3gnt6, c1galt1, c2gnt, fut2 and st7gal1. No changes were observed in Core2‐GlcNAc‐Transferase c2GnT3. Germ free mouse colonoids express enterocytes, goblet cells, and enteroendocrine cells, similar to native tissue. Microinjection of B. dentium secreted factors into colonoids derived from germ free mice resulted in increased Muc2 production, corresponding to our in vivo findings.ConclusionsThis work illustrates that the human commensal B. dentium is able to modulate the mucus profile in the colon. As B. dentium is a key colonizer of the infant gut, we speculate that this may be particularly important in the development of a mature mucus barrier and may have effects later in life.