bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antioxidant activity of the seed oil extract from Eryngium carlinae “in vitro” and in Saccharomyces cerevisiae
Authors: Mejía‐Barajas Jorge A. , Rodríguez‐Lino Adriana Lizbeth , Torres‐Martínez Rafael , Salgado‐Garciglia Rafael , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo 
Published: November 2023

Free radicals are unstable and reactive species, that modified molecules as carbohidrates, lipids, proteins or genetic material producing oxidative stress. A number of pathologies has been associated to this uncontrolled reactions. The importance to study new possibilities against the imbalance in organisms concern with antioxidants which are molecules that prevent or slow molecules oxidation. Eryngium carlinae is a plant with medicinal properties. A number of techniques has been developed to study antioxidant properties, as in vitro or using Saccharomyces cerevisiae yeast as biological model. We obtained the seed oil extract from Eryngium carlinae by Soxhlet method and evaluated its antioxidant capacity. Results obtained showed a higher antioxidant activity of seed oil from Eryngium carlinae than ascorbic acid as compared.Support or Funding InformationCONACYT 169093 to ASM; CIC‐UMSNH 2.16 to ASM
Engineered Caspases with Altered Specificities Enable Identification of Exosites
Authors: Hardy Jeanne A , Hill Maureen , MacPherson Derek J , Wu Peng , Julien Olivier , Wells James A 
Published: November 2023

Exosites for substrate binding are promising for development of inhibitors and activators with novel mechanisms of actions. Despite their potential, to date there is no systematic way to identify exosites on enzymes. We have developed a method for identifying exosites, and applied it to an important class of proteases, the caspases. To identify exosites, we built a version of caspase‐7 that maintained all exosites, but that has the active‐site specificity of caspase‐6. This new caspase allows us to categorize substrates as being active‐site or exosite driven. Simple substitution of the critical active‐site residues from caspase‐6 dramatically diminished caspase‐7 activity, but did not alter specificity, indicating that a more holistic evolutionary procedure would be needed to achieved the desired hybrid caspase. Using our GFP‐based reporter of caspase activity (CA‐GFP), we implemented a flow‐cytometry based directed evolution screen for caspase‐7 variants with caspase‐6 cleavage patterns. The caspase‐7 variants with the most strongly altered specificity displayed kinetic values similar to wild‐type caspase‐6 and mimicked caspase‐6 cleavage patterns with protein substrates. Our seven crystal structures of evolved caspases bound to either DEVD or VEID substrates allowed us to observe how the new residues in the active site contributed to the change in specificity by altering the conformation of a critical active site loop. Using N‐terminomics on the entire human proteome, we observed that the substrate specificity of the evolved caspase was superimposible with caspase‐6 and unlike the parent scaffold, caspase‐7, suggesting that our directed evolution was successful. This allowed us to identify Lamin A/C as an exosite dependent substrate. Ultimately we hope that this approach will identify exosites for recognition of just a subset of key regulatory or disease‐causing substrates, which will lead to more specific small‐molecule modulators of caspase function.Support or Funding InformationThis work was supported by the National Institutes of Health (GM80532) to JAH and (GM081051) to JAW.
Late endosomes and its unique anionic lipid bis(monoacylglycero)phosphate act as doorways for the endosomal escape and cytosolic entry of the fluorescently‐labeled dimeric cell‐penetrating peptide dfTAT and bioactive cargos
Authors: Erazo‐Oliveras Alfredo , Najjar Kristina , Truong Dat , Wang Ting‐Yi , Pellois Jean‐Philippe 
Published: November 2023

One of the key challenges and a multibillion dollar issue in the field of delivery of biologics/therapeutics inside live cells is endosomal entrapment. Currently, there is an urgent need to define the mechanisms of cell penetration and endosomal escape at a cellular and molecular level. A better understanding of how cell penetration and endosomal escape occurs will allow the improvement of existing delivery strategies. Our laboratory has shown that dfTAT, a recently identified delivery tool, can circumvent this giant hurdle. In this work we describe the use of dfTAT, a dimeric fluorescently‐labeled peptide found to promote endosomal leakage extremely efficiently and without deleterious effects to cells1, as a model cell‐penetrating peptide that can reveal the cellular organelles and factors involved in endosomal escape. By controlling/fine‐tuning the trafficking of dfTAT within the endocytic pathway, we have identified late endosomes as the unique organelles rendered leaky by dfTAT and as the exit point for dfTAT endosomal escape. Following endocytic uptake and trafficking, dfTAT binds bis(monoacylglycero)phosphate (BMP), a negatively‐charged phospholipid enriched in late endosomes and, consequently, causes endosomal leakage in cellulo. Moreover, dfTAT binds and causes leakage of bilayers containing BMP but not bilayers containing two other negatively‐charged lipids, phosphatidic acid (PA) and phosphatydilglycerol (PG), in vitro (PG being a structural isomer of BMP). Finally, we establish that an anti‐BMP antibody is capable of blocking leakage in vitro and endosomal escape in cellulo. Together, these data not only identify late endosomes as the cellular gateways utilizable for efficient delivery protocols, but also sheds light on a mechanism that may provide a fundamental basis for the rational design of future cell‐permeable molecules that are highly active yet display low cytotoxicity.Support or Funding InformationRO1GM087227, R01GM087981 and RO1GM110137
Mutagenesis Study to Reveal Important Determinants of XXT2 Catalytic Activity
Authors: Smith Adrienne Leah , Culbertson Alan T. , Zabotina Olga A. 
Published: November 2023

Xyloglucan (XyG) is the main hemicellulosic component of the primary cell wall in eudicots and nongraminaceous monocots. Three xyloglucan xylosyltransferases, including XXT2, are known to encode XyG xylosyltransferases, key enzymes in the process of XyG biosynthesis.A computational model of XXT2 was used to identify amino acids, F204, K207, D228, S229, D230, H378, putatively localized in the active site of XXT2 and mutagenesis study was performed to confirm predictions. All created mutants were expressed in E. coli SoluBL21 in pET20b plasmids containing an N‐terminal GB1 fusion tag. To increase yield and purity for future biophysical experiments, such as isothermal titration calorimetry and circular dichroism, the mutated genes were amplified from the pET20b plasmids, ligated into pGen2 plasmids and transfected into HEK293 cells. Results of the enzyme assay demonstrated that K207, D228, D230, and H378 are critical for enzyme activity due to their proposed coordination of the divalent cation. Reduced activity seen for the mutation made at F204 suggests that while this site is important for enzyme activity, it is not as crucial as the other sites tested. In addition, cysteine residues were selected for mutagenesis to investigate their effect on disulfide bond formation shown to be involved in hetero‐ and homo‐ dimer formations. Expression and analysis of these mutants is currently in process.Truncations of XXT2 from both the N‐ and C‐ends were used to examine the contributions of these termini to protein solubility and activity. In N‐terminal truncations longer than 31 residues, both protein expression and activity of XXT2 were negatively affected, indicating a possible effect on protein folding. Increasing sizes of truncations from the C‐terminus showed an increase in protein expression but a decrease in its catalytic activity. We conclude that the C‐terminus is not involved in protein folding but rather substrate binding. This truncation identified a series of six positively charged residues between 24 and 44 amino acids from the C‐terminus that may play a role in substrate binding. Mutagenesis of these residues revealed that four of the mutants have reduced catalytic activity.We conclude that the N‐terminal of XXT2 is involved in protein folding while the C‐terminal is involved in substrate binding. Furthermore, active site residues K207, D228, D230 and H378 are proved to be critical for enzymatic activity of XXT2.Support or Funding InformationThis research is supported by NSF‐MCB Grant #1121163
Structure and biosynthesis of complex N‐glycan cores and antennae in nematodes
Authors: Wilson Iain B. H. , Yan Shi , Paschinger Katharina 
Published: November 2023

There are thousands of nematode species, some of which are parasites such as Ascaris suum, Trichuris suis or Haemonchus contortus; others such as the popular model organism, Caenorhabditis elegans, are free‐living. Although all are animals, they lack some of the characteristics of glycosylation found in vertebrates, but their ‘foreign’ nature is relevant to the interactions of nematode parasites with their hosts. The presence of complex modifications of the chitobiose core region of nematode N‐glycans and of phosphorylcholine on the glycan antennae are typical, but the details are species‐specific. Whereas Caenorhabditis N‐glycans can contain up to four fucose residues, this number is reduced in parasitic species. Also galactosylation, at unusual positions, is a recurrent theme. Of the three core fucosyltransferases and one fucose‐modifying galactosyltransferase known, unusual and unexpected enzyme specificities have been revealed by studying both enzyme specificities and the effect on the glycome of deleting the relevant genes; even the processing hexosaminidases of nematodes are only distantly related to their ‘cousins’ in other invertebrates which are responsible for formation of paucimannosidic glycans. Thereby, glycomics, genetics and biochemistry are not only important tools for understanding nematode glycan biosynthesis, but open the way to identifying carbohydrate motifs which play roles in the interactions of these organisms with their hosts.Support or Funding InformationAustrian Science Fund (FWF) grants P21946 to K.P. and P23922 to I.W.
Bacterial glycosidases acting on red blood cells
Authors: Bourne Yves , Ponchel Guillaume , Roig‐Zamboni Véronique , Bennett Eric P , Henrissat Bernard , Clausen Henrik , Sulzenbacher Gerlind 
Published: November 2023

Glycosidases represent the major catalytic machinery for the breakage of glycosidic bonds. A classification system for glycosyl hydrolases, based on sequence similarity, is available on the dedicated carbohydrate‐active enzymes (CAZy) database and has led to the definition of more than 130 different families (1). Among them, two families of bacterial glycosyl hydrolases, identified by a combination of screening and bioinformatics approaches, perform efficiently and specifically in conversion of red blood cells for the production of universal red blood cells (2,3). Most of the enzymes are found in symbiotic or soil‐borne bacteria. Because members of one family have essentially identical kinetic properties with both branched and linear substrates, they can also act in the enzymatic removal of immunodominant alpha3Gal xenotransplantation epitope (3). We will describe the molecular determinants responsible for the fine substrate specificity of these enzymes and emphasize the potential biomedical applications in remodeling glycan structures.Support or Funding InformationSupported by the CNRS and DGA
Silencing Mannosylphospho Dolichol Synthase with shRNA Impacts Differentiation of Capillary Endothelial Cells
Authors: Baksi Krishna , Zhang Zhenbo , Banerjee Aditi , Serrano Jesus E , Perez Linyer E , Linares Lorraine , Seijo Arelis , Sanchez Neysharie , Katiyar Usha , Banerjee Dipak K 
Published: November 2023

Mannosylphospho dolichol synthase (DPMS) is down stream of N‐acetylglucosaminyl 1‐phosphate transferase (GPT) in the dolichol pathway of asparagine‐linked (N‐linked) protein glycosylation. When capillary endothelial cells are treated with a GPT inhibitor tunicamycin, angiogenesis is inhibited quantitatively reducing the breast tumor growth in nude mice by inducing unfolded protein response (upr)‐mediated apoptosis. There was also a quantitative loss of DPMS catalytic activity. We, therefore, hypothesized that there is a cross‐talk between the GPT and DPMS, and DPMS independently plays a significant role in angiogenesis. To evaluate, we have isolated a stable capillary endothelial cell line in which DPMS has been knocked down after transfecting the cells with DPMS shRNA plasmid in pSilencer 4.1‐CMV neo vector. The control used a scrambled shRNA sequence. DPMS expression in each cell clone was monitored by measuring catalytic activity, qRT‐PCR for DPMS gene expression, western blotting and immunofluorescence microscopy for DPMS protein expression. They all have down regulated in the knockdown clone. Western blotting with Concanavalin A also indicated the presence of reduced level of N‐linked glycoproteins with terminally linked mannose. Interestingly, when the cellular morphology was followed as a function of time, the DPMS knockdown clone exhibited lumen‐like structure formation more frequently than the control. Clonogenic assay indicated 0% survival of DPMS knockdown cells after 48 hours of tunicamycin (1 μg/mL) treatment. We, therefore, conclude that DPMS silencing makes cells less angiogenic, more susceptible to the anti‐angiogenic glycotherapy tunicamycin and importantly impacts the differentiation characteristics of the cell.Support or Funding Information).Supported in part by grants from NIH/NIMHD 2G12MD007583 (KB) and NSF‐EPSCoR RII Track 1 Grant # EPS‐1002410 (DKB)
Elevated O‐GlcNAcylation levels improve mitochondrial function
Authors: Tan Ee Phie , Swerdlow Russell , Slawson Chad 
Published: November 2023

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a highly dynamic post‐translational modification that involves an addition of a single N‐acetylglucosamine to serine and threonine residue in mitochondria, nuclear and cytoplasmic proteins. O‐GlcNAc cycling is the addition and removal of O‐GlcNAc by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) respectively. Disruptions in O‐GlcNAc cycling contribute to diseases such as diabetes, cancer, and neurodegeneration. Accumulative dysfunctional mitochondria can also lead to the development of neurodegenerative diseases, and importantly, O‐GlcNAcylation regulates mitochondrial function. Previously, using a proteomics based screen we found that altering the expression of OGT and OGA disrupted mitochondrial protein expression, including proteins involved in the respiratory chain and TCA cycle. Furthermore, mitochondrial morphology in the over‐expressing cells had reduced and disorganized cristae and altered shape and size. Both cellular respiration and glycolysis was impaired in the OGT/OGA gain of function cells. These data suggested that O‐GlcNAc cycling was essential for the proper regulation of mitochondrial function. We hypothesized that prolonged elevations in cellular O‐GlcNAc levels would alter the metabolic profile of the cell. We disrupted O‐GlcNAc cycling by either treating SH‐SY5Y neuroblastoma cells with low levels of Glucosamine (GlcN) or OGA inhibitor Thiamet‐G (TMG). Importantly, the metabolic substrate for O‐GlcNAcylation is UDP‐GlcNAc. UDP‐GlcNAc is synthesized via the hexosamine biosynthetic pathway, which is highly sensitive towards the availability of nutrients in the cellular environment. GlcN feeds into the hexosamine biosynthetic pathway after the first rate‐limiting enzyme GFAT1, glutamine fructose‐6‐phosphate aminotransferase 1, and additional supplementation of GlcN increases O‐GlcNAcylation via elevating the synthesis of UDP‐GlcNAc. We found that prolonged TMG/GlcN treatment improved mitochondrial function. In the TMG/GlcN treated cells, reactive oxygen species (ROS) production was significantly reduced. Interestingly, the protein expression of the mitochondrial antioxidant, manganese superoxide dismustase (MnSOD) was lower. Additionally, ATP production was lower in these TMG/GlcN treated cells. Furthermore, we found that mitochondrial were longer in shape and mitochondrial fusion/fission protein expressions were decreased. Altogether, these data demonstrated that prolonged alterations to the cellular homeostasis of O‐GlcNAc levels improve mitochondrial function likely through reprogramming of metabolic transcriptional programs. Further understanding of how O‐GlcNAc cycling regulates metabolism will provide new insights into metabolic diseases such as cancer and Alzheimer's.Support or Funding InformationRO1DK100595
O‐GlcNAcylation of the Human Kinome
Authors: Liu Xin , Han Guanghui , Wells Lance , Graves Lee , Zhu Heng , Neiswinger Johnathan , Hart Gerald W. 
Published: November 2023

O‐GlcNAc, first characterized more than 30 years ago, is an O‐linked β‐N‐acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residues. O‐GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O‐GlcNAc to proteins is catalyzed by O‐GlcNAc transferase (OGT), while its removal is catalyzed by O‐linked N‐acetyl‐β‐D‐glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein‐protein interactions. O‐GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders.Protein phosphorylation is a post‐translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals. The regulation of phosphorylation is a central mechanism in cell health and disease. O‐GlcNAcylation has extensive interplay with phosphorylation at the site level on proteins and by modifying kinases. However, the roles of O‐GlcNAcylation have only been studied on a few kinases.In vitro O‐GlcNAcylation of large kinase microarrays indicates that well over one‐half of all human protein kinases are substrates for OGT. Multiplexed kinase inhibitor beads (MIBs) are being used to confirm the O‐GlcNAcylation of kinases in vivo. These affinity beads have enriched at‐least 117 kinases as determined by mass spectrometry and HCD and ETD fragmentation are being used to map their O‐GlcNAcylation sites.Support or Funding InformationSupported by NIH P01HL107153. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU
Heterologous Expression of Multidrug Resistance Proteins
Authors: Marchesseault Collette A. , Vogel Pia D. , Wise John G. 
Published: November 2023

One key to developing effective cancer treatments for many patients today involves the inhibition of multidrug resistance pumps overexpressed in cancer cells. These membrane proteins in the ABC transporter family act to remove the chemotherapeutics from the cell, rendering normal chemotherapies ineffective. Two ABC transporters that are especially problematic and that cause multidrug resistances are P‐glycoprotein (P‐gp) and the Breast Cancer Resistance Protein (BCRP). High‐throughput computer modeling was used to narrow the search for possible inhibitors of P‐gp and many of these inhibitors have been shown to be effective in biochemical assays (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular pharmacology, 86(6), 716–726) as well as in reversing multidrug resistances in human cell culture assays (Follit, C. A., Brewer, F. K., Wise, J. G., & Vogel, P. D. (2015) Pharmacology Research & Perspectives, 3(5) e00170, doi:10.1002/prp2.170). In order to expedite the production of P‐gp protein for inhibitor testing, structural and mechanistic studies, new bacterial expression systems are being developed. It is hypothesized that such systems would allow for an increase in protein yield and the overall efficiency of the isolation of the transporter protein. It should be noted that glycosylation of P‐gp is not essential for its folding or activity. To accomplish these aims, P‐gp was cloned into E. coli behind the glpF glycerol transporter as a fusion gene. GlpF is a native E. coli membrane protein known to be expressed at high levels and was included to aid with the expression, folding and insertion of P‐glycoprotein into the bacterial membrane. Clones were verified by DNA sequencing and protein expression was examined using Western blot analysis using anti‐His6 and anti‐P‐gp antibodies to screen for the presence of P‐gp. Purified sub‐bacterial particles and isolated transporter preparations were assayed for ATP hydrolysis by a coupled enzyme assay to assess catalytic activity. Positive preliminary results for the expression of protein in E. coli were obtained when the bacteria were grown in the presence of a known inhibitor of P‐gp. A similar bacterial expression has also been used for BCRP expression. Gene block assembly was used to synthesize a BCRP gene which was then verified by DNA sequencing. Preliminary results suggest that expression of BCRP in E. coli membranes has also been accomplished. Optimization of the growth and purification of P‐gp and BCRP from E. coli cells will be discussed.Support or Funding InformationThis work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, Hamilton Undergraduate Research Scholarship, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, New England Biolabs / Lynn McBee, and a private gift from Ms. Suzann Ruff of Dallas, Texas.