bluuebunny/crossref_metadata_2025_split huggingface dataset:
In pursuit of mammalian polyamine oxidase crystal structure (774.4)
Authors: Zamora Diana , T. Melendez Jose , Galaleldeen Ahmad 
Published: June 2021

Mouse Polyamine Oxidase (mPAO) is a mammalian flavoprotein that is necessary for the catabolism of polyamines. mPAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine respectively. It is known that normal levels of polyamines are important for cell growth, yet their actual function is not well understood. The structure of mammalian PAO has yet to be determined; however structures for maize and yeast PAO (Fms1) exist. Although the identity between mPAO, Fms1, and maize PAO is only 20%, it has been shown, through sequence comparison and mutational analysis, that a conserved histidine residue at position 67 of Fms1 and 64 of mPAO helps to properly position the amine substrate for oxidation. Determining the crystal structure of mPAO will help identify other residues that play a role in substrate binding and catalysis.mPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. mPAO was expressed and purified using a Nickel column followed by an anion exchange column. Following the purification process, analytical ultracentrifugation sedimentation velocity experiments were performed to determine the oligomerization state and homogeneity of the protein.The sedimentation velocity experiments revealed a homogeneous, monomeric mPAO in solution, in contrast to the dimeric form of Fms1 in solution. Crystallization trials are underway to determine the structure of mPAO.
Protein function prediction using ProMOL and PyMOL (777.3)
Authors: McKay Talia , Hart Kaitlin , Bernstein Herbert , Tedla‐Boyd Weinishet , Craig Paul 
Published: June 2021

ProMOL, a plugin for the molecular graphics system PyMOL, was designed to characterize protein structures that lack functional annotation; there are currently over 3700 PDB entries with “unknown” functions. ProMOL was created, and is constantly being revised, by members of the RIT and Dowling College communities. ProMOL compares query protein structures against a library of active site motif templates, calculates Levenshtein distances and RMSD values, and generates a 3D visual alignment between query and motif template. The best results are then submitted to BLAST, Dali and Pfam for analysis. If the data produced by these four bioinformatics programs lead to a preliminary conclusion concerning the function of an uncharacterized structure, the behavior of the protein is then tested in vitro. To date, we have found >90 significant “hits” for uncharacterized protein structures using ProMOL’s motif library. These structures are currently being analyzed via BLAST, Dali, and Pfam. To date the most promising alignments include PDB entries 2HRZ (proposed as isomerase/NAD‐dependent epimerase), 4EZI (confirmed as an esterase; see the related poster by Kaitlin Hart), 3L1W (proposed as a nuclease), and 2O14 (proposed as a hydrolase/aminopeptidase). This work has been supported in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1.Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1
Metallomics and proteins expression of diabetic rats (778.2)
Authors: Braga Camila , Vieira José , Moraes Paula , Cavecci Bruna , Fernandes Ana Angélica , Okoshi Marina , Padilha Pedro 
Published: June 2021

The aim of this study was to analyze changes in the expression of proteins and compare metal ions (Cu, Zn, Se= using atomic absorption spectrometry with flame and Mg= using atomic absorption spectrometry with oven graphite) in plasma gels (two‐dimensional electrophoresis, pool, eight replicates/group, strips with pH 4‐7, polyacrylamide gels at 10% w/v) of normal (G1), diabetic (G2) and diabetic treated with insulin (G3) rats. In the context, 251 spots were found in G1, 300 in G2 and 227 in G3 (percentual matching of 77, 84 and 85%. The differential analysis indicated difference in expression (p<0.05‐ ANOVA) of 13 (G1xG2), 10 (G1xG3) and 18 (G2xG3) spots when groups were compared. The qualitative determination of metal ions of interest indicated to G1, G2 and G3 that 87 (48.60%), 154 (75.49%), 48 (23.36%) contained Cu; 33 (18.43%), 47 (23.03%), 141 (71.57%) Mg; 148 (82.68%), 108 (52.94%), 155 (78.68%) Se and 19 (10.61%), 7 (3.43%), 69 (35.02%) Zn. Observed difference in the presence of ions in the same protein in relation to different groups, and the next is to understand how these ions are coordinated to protein.Grant Funding Source: Financial Support: Fapesp 2011/21672‐1
A novel dynamic delivery system enabling high efficiency transfection of cells (779.4)
Authors: Ludtke James , Lauer Anthony , Storck Austin , Rossi Nicholas , Neder Karen , Pinchuk Anatoly , Juckem Laura 
Published: June 2021

Transfection is a technique to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity. To address these shortcomings, Mirus Bio has developed a novel non‐liposomal technology called the TransIT‐X2™ Dynamic Delivery System. This unique polymeric delivery system is able to condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm.In this study, we demonstrate functional transfection of labeled plasmid DNA co‐delivered with labeled siRNA illustrating the dual abilities of the system. Additionally, extensive head‐to‐head comparisons with Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT‐X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2‐fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT‐PCR. miRNA function was assessed by the decrease in the miR‐1 target mRNA PTK9. TransIT‐X2 shows effective knockdown via siRNA or miRNA.The TransIT‐X2 Dynamic Delivery System offers researchers exceptional broad‐spectrum transfection of plasmid DNA and siRNA. This cutting edge, non‐liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies.
Cytosolic calcium regulates nanoparticle egress from alveolar epithelial cells (780.11)
Authors: Sipos Arnold , Chow Robert , Borok Zea , Kim Kwang‐Jin , Crandall Edward 
Published: June 2021

We previously reported that polystyrene nanoparticles (PNP) translocate across alveolar epithelium. In this study, we investigated PNP exit from alveolar epithelial cells (AEC) using confocal live‐cell imaging. Primary type I cell‐like rat AEC monolayers on Anopore filters were apically exposed to 80 μg/mL PNP (20 nm, carboxyl‐modified, near‐infrared labeled) for 12 h, followed by washing and measuring cytoplasmic [PNP] as a function of time, in the presence/absence of agents that elevate (e.g., ATP) or chelate (e.g., BAPTA‐AM) intracellular [Ca2+] ([Ca2+]i), as confirmed with fluo‐4, and/or nocodazole (Noc), an inhibitor of microtubule polymerization. Results show that cytoplasmic PNP both co‐localize with lysotracker‐positive endosomes and exit cells in two stages: initial rapid egress for ~2 h, followed by slower linear egress. Overall t0.5 for egress is 1.5 h, much faster than that for apical PNP uptake. ATP increases [Ca2+]i and egress rate (to t0.5 ~0.5 h), while BAPTA prevents increased [Ca2+]i and egress rate after ATP exposure. Noc, with or without ATP, inhibits PNP exit via the rapid initial egress process. These data suggest that: (1) PNP egress rate is dependent on [Ca2+]i, (2) PNP uptake is rate‐limiting for transepithelial PNP translocation, (3) PNP egress occurs via both rapid lysosomal exocytosis and slow ‘diffusion’‐like process(es), and (4) kinetics of egress of PNP from AEC can be modulated.Grant Funding Source: NIH; Hastings & Whittier Foundations
Functionalizing silica‐coated iron oxide nanoparticles for imaging and targeted cancer therapeutics (780.3)
Authors: Nagalla Raji , Liao Zi Wei , Webb Andrew , Flynn Nolan , Kolodny Nancy 
Published: June 2021

This project focuses on the surface modification of silica‐coated superparamagnetic iron oxide NPs to achieve tissue specific drug delivery to pancreatic tumors. The treatment of pancreatic cancer remains a challenge in the biomedical community. Over half of cases are diagnosed after the cancer has spread to other tissues, and in these cases the 5‐year survival rate is 2%. The non‐specific nature of currently available chemotherapies has spurred interest in developing vehicles to deliver potent therapies specifically to tumors. Nanoparticles (NPs) present an ideal vehicle due to their high surface area, small size, and low toxicity. Recent work explored the conjugation of NPs to a targeting moiety: monoclonal antibody CHO 31.1, which recognizes gpA33, a membrane glycoprotein overexpressed in 50% of pancreatic cancers and 95% of colorectal cancers. Building on existing methods, the antibody was attached to the silica surface of the NPs via a polyethylene glycol (PEG) linker. In vitro techniques were also developed to quantify NP detection using magnetic resonance imaging (MRI). Attachment of multiple moieties was explored using orthogonal surface chemistries. Cellular internalization was determined using confocal microscopy. Moving forward these novel nanoparticles will be further characterized for morphology, size and zeta potential, and for their utility as contrast agents for tracking in vivo.Grant Funding Source: Supported by NIH grant #1R15CA156393‐01A1
In vitro reconstitution of lipid‐dependent membrane protein topological switching (785.1)
Authors: Vitrac Heidi , Bogdanov Mikhail , Dowhan William 
Published: June 2021

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. Systematic alteration of lipid membrane composition uncovered a role for lipid‐protein interactions in determining dynamic post‐assembly changes in and initial topogenesis of membrane proteins. Using an in vitro proteoliposome system in which lipid composition can be systematically controlled not only before (liposomes) but also after (fliposomes) reconstitution, we have determined the minimum and sufficient requirements for lactose permease (LacY) to flip between topologically distinct states. Changes in phosphatidylethanolamine levels after final protein folding resulted in re‐orientation of the original conformer mixture, establishing a dynamic reversibility in topology without molecular chaperone or translocon involvement. Using Förster resonance energy transfer approaches, we further investigated the kinetics of lipid exchange and LacY flipping. The rates of protein flipping in both directions were on a second scale and occurred almost instantaneously as the lipid composition of the proteoliposomes changed. These observations demonstrate a potential thermodynamically driven lipid‐dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within cells independent of other cellular factors.Grant Funding Source: Supported by NIH grant GM R37 20478
Oligomerization of Hsp70 upon insertion into lipid membranes (785.2)
Authors: Armijo Gabrielle , Cauvi David , Okerblom Jonathan , Lopez Victor , Gonzales Derek , Arispe Nelson , De Maio Antonio 
Published: June 2021

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER‐Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. We propose that membrane interaction is the first step in the export process of Hsp70, the major inducible hsp, since this protein has been detected on the surface of stressed cells. The question that emerges is how does this highly charged cytosolic protein interact with lipid membranes? Using a liposome insertion assay, we showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments, and the region embedded into the lipid membrane was mapped to the C‐terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted across a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.Grant Funding Source: Supported by NIH R01 GM098455
Unraveling mTOR signaling: from two complexes to nuclear dynamics (786.1)
Authors: Sarbassov Dos 
Published: June 2021

The protein kinase mTOR is a central component of the essential and highly conserved nutrient‐sensing pathway. Our biochemical characterization of mTOR led to discovery of a novel nuclear envelope mTOR complex that is distinct from the known complexes of mTOR (mTORC1 and mTORC2) by size and also by the sub‐cellular localization. We found that mTOR in association with the nuclear pore component forms the nuclear envelope complex that regulates the nutrient‐dependent nuclear import of ribosomal proteins. We found that similar to the amino acid deprivation, the mTOR kinase inhibitors caused a substantial decrease in abundance of ribosomal proteins in the nuclear but not cytosolic fraction. The mTOR‐dependent nuclear import of ribosomal proteins is also supported by analysis of the GFP‐tagged ribosomal protein in the cell based imaging assays. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTORC1 by rapamycin or loss of mTORC2. Thus, our study identified a novel role of mTOR in regulation of the nuclear import of ribosomal proteins that might play a role in ribosomal biogenesis and contribute to the nutrient‐dependent regulation of cell growth.Grant Funding Source: Supported by Texas State (CPRIT)
Inhibiting epidermal growth factor receptor signal transduction via metabolic oligosaccharide engineering sensitizes pancreatic cancer cells to drugs (788.4)
Authors: Mathew Mohit , Tan Elaine , Saeui Christopher , Bovonratwet Patawut , Bhattacharya, Rahul , Yarema Kevin 
Published: June 2021

Metabolic Oligosaccharide Engineering is a versatile technique that can be used to change patterns of glycosylation by altering the availability and abundance of biosynthetic precursors of glycans; for example, 1,3,4‐O‐Bu3ManNAc can be used to increase flux through the sialic acid biosynthetic pathway. Because glycosylation is a non‐template driven process, such an increase in the levels of sialic acid results in the favoring of increasingly sialylated glycoforms. In a previous study we found that 1,3,4‐O‐Bu3ManNAc increased the sialylation of dozens of N‐linked glycoproteins in SW1990 pancreatic cancer cells including Epidermal Growth Factor Receptor (EGFR) by ~2.6 fold. EGFR is known to play important roles in cancer progression but relatively little is known about how alterations to the glycosylation of this protein affect its behavior. Therefore we found it interesting that the 1,3,4‐O‐Bu3ManNAc‐driven increase in EGFR sialylation inhibited signal transduction of this receptor beginning at the level of phosphorylation and extending downstream to the transcriptional level. We reasoned that these cancer cells would have a decreased capacity for EGFR signaling and be more susceptible to drugs such as Erlotonib and Gefitinib that target EGFR phosphorylation. This premise was confirmed experimentally, with combination indexes of ~0.3 showing strong synergy between 1,3,4‐O‐Bu3ManNAc and either drug. Further studies using additional approaches (e.g., non‐natural ManNAc analogs or modulation of glycolytic flux) also show synergy in preliminary experiments, opening the door to multi‐pronged approaches that combine the benefits of glycosylation‐induced EGFR signal suppression and synergistic interactions with existing cancer therapeutics.Grant Funding Source: Supported by R01CA112314
Complex N‐linked glycans serve as a determinant for exosome/microvesicle cargo recruitment (788.5)
Authors: Liang Yaxuan , Eng William , Colquhoun David , Dinglasan Rhoel , Graham David , Mahal Lara 
Published: June 2021

Exosomes, also known as microvesicles (EMVs), are nano‐sized membrane particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell‐cell signaling, immune activation and suppression, and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein cargo, which can dictate pathogenicity. While some mechanisms have been proposed for the regulation of EMV cargo selection, little attention has been paid to N‐linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from the overall cellular membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we explore further the role of glycosylation in EMV cargo selection. We identify specific glycosylated targets and associated proteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Further, we show that genetic manipulation of the glycosylation levels of a MV cargo glycoprotein EWI‐2 directly impacts its recruitment as a function of N‐linked glycan sites. Taken together, our data provided strong evidence that N‐linked glycosylation directs glycoprotein sorting into EMVs and serves as a determinant of EMV cargo selection.Grant Funding Source: Supported by Mizutani Foundation for Glycoscience
O‐GlcNAc occurs in the protozoan parasite Toxoplasma gondii and is part of the histone code (790.4)
Authors: Silmon de Monerri Natalie , Nardelli Sheila , Calloway Myrasol , Nieves Edward , Angeletti Ruth , Weiss Louis , Wu Peng , Kim Kami 
Published: June 2021

O‐GlcNAcylation, the addition of N‐acetylglucosamine to Serine or Threonine residues, is an abundant, dynamic posttranslational modification that occurs in many eukaryotes; very little is known about its role in early eukaryotes including protozoa. Here, we describe O‐GlcNAcylation in the protozoan parasite Toxoplasma gondii, which causes one of the most common parasitic infections in humans and warm‐blooded animals, Toxoplasmosis. T. gondii is an obligate intracellular parasite, which has the unique ability of being able to switch between two developmental stages, a phenomenon that is crucial for pathogenesis and is thought to be regulated by sensing of nutrient availability and metabolism. How parasites sense changes in the host cell environment is unclear; since O‐GlcNAc is important for metabolic sensing in other eukaryotes, this led us to hypothesise that O‐GlcNAc is a metabolic sensor in T. gondii, regulating developmental programming. Here, we demonstrate that both the T. gondii homologue of O‐GlcNAc transferase (TgOGT) and O‐GlcNAc are present in nuclear and cytoplasmic compartments in T. gondii parasites. As O‐GlcNAc is an important epigenetic regulator, we focussed on its role on T. gondii histones, on which we show by mass spectrometry and immunoprecipitation that it is also present, indicating that it is a dynamic modification in T. gondii. To identify more O‐GlcNAcylated proteins associated with chromatin, we performed immunoprecipitations and mass spectrometry. Surprisingly, native ChIP‐chip experiments localised O‐GlcNAc to centromeric and telomeric regions of the T. gondii genome, where it colocalises with other epigenetic modifiers, suggesting that it plays a role in mitotic chromatin dynamics. Current efforts include overexpression of dominant negative mutants and conditional knockdown to investigate the function of TgOGT in T. gondii.Grant Funding Source: R21AI101801‐01A1
Complexation of phospholipase C isozymes regulate cytosolic and nuclear signaling and insulin secretion (796.14)
Authors: Cline Gary 
Published: June 2021

Insulin secretion from the pancreatic islet β‐cell is stimulated not only by glucose, but also by binding of acetylcholine to the M3 muscarinic receptor. Acetylcholine binding activates PLCβ2. Postpandrial increases in glucose metabolism by the β‐cell lead to Ca2+ influx and activates PLCδ1. Thus, the β‐cell has redundant mechanisms to increase the provision of PLC‐derived second messengers of insulin secretion. PLCβ2 and PLCδ1 isozymes can exist in an enzymatically‐inactive complexed state that is dissociated by either cholinergic agonists or by elevated glucose. Dissociation of the PLCβ‐δ complex enables PLC enzymatic activity.Our Co‐IP studies indicate that the PLCβ‐δ complex exists in the INS‐1 β‐cell line, and that treatment with muscarinic agonist, carbachol, led to dissociation of PLCβ2 from PLCδ1. Spatial localization of PLC isozymes was evaluated with indirect immunofluorescence microscopy. At basal conditions, PLCβ2 and PLCδ1 were colocalized throughout the cytosol, but were excluded from the nucleus. Dissociation of the PLCβ‐δ complex occurred with carbachol or high glucose, and notably a pronounced translocation of the PLCδ1 to the nucleus occurred.These data are the first showing that 1.) PLCβ2 and PLCδ1 exist in a complexed state in β‐cell lines, 2.) PLCβ‐δ decomplexation occurs upon cholinergic or glucose stimulation, and 3.) PLCδ1 translocates to the nucleus. Intriguingly, dissociation and activation of the PLC isozymes has the potential to generate both cytosolic and nuclear signaling intermediates. These studies are timely in light of the recent appreciation of the paracrine role of acetylcholine release from islet α‐cells, and the association between variants in human muscarinic receptor gene and increased risk of type 2 diabetes.