bluuebunny/crossref_metadata_2025 huggingface dataset:
Efforts towards crystallization of Taq DNA polymerase mutants (967.4)
Authors: Valencia Alfredo , Kroll Jacqueline , Sazinsky Matthew , Leconte Aaron
Published: June 2021

Taq DNA polymerase is an essential enzyme in biotechnological applications like the polymerase chain reaction (PCR) and Sanger sequencing. Within the last two decades, several groups have identified mutant Taq with the unnatural ability of incorporating 2’‐modified DNA, which holds implications in its use for future applications, but the origins of this activity are currently unclear. The immediate goal of this project is to develop a generalizable method for cloning, expressing and purifying Taq DNA polymerases to move forward with their crystallization and x‐ray crystal structure determination. A cloning procedure to remove undesired linker sequences present for the wt and mutant Taq plasmids has been successfully developed, and expression of these plasmids in a BL21 cell line of E. coli has been performed. The purification of enzymes by polyethyleneimine (PEI) precipitation and fast pressure liquid chromatography (FPLC) is currently being optimized. Finalizing these procedures will provide the groundwork for using x‐ray crystallography to determine and compare the structures of wild type (wt) and mutant Taq while incorporating modified and unmodified nucleotides, which in turn will aid in future protein‐engineering efforts.
It takes two to tango: fine‐tuning of soluble guanylate cyclase activity via protein‐protein interactions and conformational changes (969.3)
Authors: Garcin Elsa , Seeger Franziska , Quintyn Royston , Tanimoto Akiko , Williams Garrett , Tsutakawa Susan , Wysocki Vicki , Tainer John
Published: June 2021

Nitric oxide (NO) regulates many biological processes, including vasodilation, neurotransmission, and immune response. The primary receptor for NO is heterodimeric soluble guanylate cyclase (sGC), which is a validated target for therapeutic intervention in pulmonary arterial hypertension.

We have solved the first x‐ray structure of wild‐type heterodimeric catalytic domains from human sGC, revealing key structural determinants for heterodimerization and conformational changes needed for catalysis. Using mass spectrometry, we determined dissociation constants for heterodimeric and homodimeric catalytic domains. Surprisingly, activity measurements showed that isolated catalytic domains are far less active than full‐length sGC.

Together, our results argue that other sGC domains are crucial to promote sGC heterodimerization in vivo. We further propose an exquisite regulation mechanism whereby the catalytic activity of sGC is finely tuned by protein:protein interactions that either promote or inhibit catalytic activity. Importantly, our structural studies provide a framework to design novel sGC activators targeted at the catalytic domain.

Grant Funding Source: Supported by American Heart Association
Characterization of active compounds produced in the biotransformation of metabolites in kombucha tea (973.2)
Authors: Seehusen Katherine , Villeneuve Mallory , Mann Francis
Published: June 2021

Kombucha is a fermented sugared black tea that has been hypothesized to provide many health benefits. The exact origin of these benefits, however, is continually being investigated. This study aims to identify the various active compound produced in the biotransformation of the metabolites during the fermentation process of the tea beverage, as well as quantify functions such as antioxidant capacity. Hydrophilic and hydrophobic liquid‐liquid extractions were performed on a filtered sample of GT’s Organic Raw Kombucha® using acetonitrile and ethyl acetate. Ferric reducing/antioxidant power (FRAP) assays were run on both the Kombucha tea and the extracts to determine their antioxidant capacity. The Kombucha tea, acetonitrile extract, and ethyl acetate extract were found to have FRAP values of 146.9, 102.7, and 71.04, respectively. The high retention of FRAP in the acetonitrile extract gives evidence that a polar hydrophobic molecule is functioning as an antioxidant in the Kombucha tea. Antioxidant capacity was further tracked using a variety of chromatographic techniques including the use of silica gel and C‐18 functionalized silica flash chromatographies, as well as reverse‐phase C‐18 HPLC. These studies aim to further purify and characterize the compounds responsible for antioxidant capacity.
Dihydroxyacetone disturbs collagen fibril formation due to side chain modifications by glycation (979.1)
Authors: Liu Weixi , Dombi George , Dain Joel
Published: June 2021

Glycation is a spontaneous process typically initiated with a condensation reaction between a reducing sugar and a protein, leading to the formation of Advanced Glycation Endproducts (AGEs). The in vivo accumulation of AGEs has been shown to modify the side chains of long lived proteins such as collagen, resulting in the alteration of their functions. The objective of this study was to use a combination of analytical techniques to evaluate the effect of dihydroxyacetone (DHA), an FDA approved tanning agent, on collagen glycation and fibril formation. Fluorescence spectroscopy revealed that DHA readily glycated collagen and induced the formation of AGEs. Moreover, addition of DHA to a collagen solution disturbed fibril formation, an observation confirmed by both UV spectroscopy and transmission electron microscopy (TEM). Compared to control solutions with collagen alone, addition of DHA showed a similar extent of fibril formation, but with a much lower rate, suggesting a reversible association of DHA which was displaced during collagen self‐assembly. The extent of fibril formation of collagen pre‐incubated with DHA for 24 hours, however, was significantly lower than untreated collagen, suggesting that DHA irreversibly glycated the collagen side chains and such modification prevented the self‐assembly of collagen necessary for fibril formation. Our future studies will focus on characterizing the DHA modification of the collagen side chains and assessing the correlation between collagen fibril formation kinetics and the degree of DHA induced glycation.
Sirtuin regulation of lysine acylation targets diverse metabolic networks in hepatic mitochondria (981.3)
Authors: Rardin Matthew , He Wejuan , Sahu Alexandria , Nishida Yuya , Verdin Eric , Gibson Bradford
Published: June 2021

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Sirtuin 3 (SIRT3) and SIRT5 are NAD+‐dependent mitochondrial protein deacylases. While SIRT3 is the predominant mitochondrial deacetylase, SIRT5 has been identified as a lysine desuccinylase. Here, we use a label‐free quantitative proteomics called MS1 Filtering to characterize lysine acylation in liver mitochondria and its regulation by SIRT5 and SIRT3 using knockout mouse models. Using this approach we identified 1190 sites of lysine succinylation and 2187 sites of lysine acetylation from isolated mitochondria. Overall, we found 939 sites (~79%) were modified by both acetylation and succinylation, while 93 sites were targeted by both SIRT3 and SIRT5. Pathway analysis revealed that SIRT3 and SIRT5 are targeting similar mitochondrial pathways including β‐oxidation, branched‐chain amino acid catabolism, TCA cycle, and ketone body synthesis. Finally, we demonstrate that SIRT5 regulates succinylation of the rate‐limiting ketogenic enzyme 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 both in vivo and in vitro. Taken together, these findings establish SIRT5 and SIRT3 as global regulators of lysine acylation in mitochondria and suggest potential overlap in regulating metabolic networks.
Black spot, black death, black pearl: the tales of bacterial effectors (99.1)
Authors: Orth Kim
Published: June 2021

Research in my lab centers around elucidating the biochemical activity of virulence factors used by bacteria to manipulate eukaryotic signaling machinery. My lab approaches the analysis of host signaling pathways by tapping into the enormous reservoir of bacterial virulence factors that are used by bacterial pathogens during infection to manipulate a eukaryotic cell. Gram‐negative bacteria can use a type III secretion system (T3SS) as a transport vehicle to deliver the virulence factors from the bacterial cytosol to the target host cytosol. The bacterial pathogen Vibrio parahaemolyticus utilizes two type III secretion systems to cause death of host cells within hours. T3SS1‐mediated cell death is completely independent of apoptosis and occurs by a sequential mechanism in which relentless induction of autophagy is followed by cell rounding and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated LC3 and increases with vacuole formation. VopQ is both necessary and sufficient to induce autophagy. The T3SS2 was recently discovered to mediate invasion of host cells. We have deciphered molecular events that occur over the 6 hour invasion, intracellular survival and lysis of the host cell and how some of the effectors contribute to this pathology. Molecular mechanisms used by this pathogen will be the focus of discussion.
Dynamics of the general secretory system viewed in near‐native conditions via atomic force microscopy (993.1)
Authors: King Gavin , Sanganna Gari Raghavendar Reddy , Frey Nathan , Mao Chungfeng , Randall Linda
Published: June 2021

In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein transfer across and into membranes. Further, it is known that SecA is the ATPase of the general secretory system and it binds SecYEG to perform translocation. In so doing, SecA makes large surface area contact with the unstructured cytoplasmic loops spanning transmembrane helices 6‐7 and 8‐9 of SecY. Despite their functional significance, measurements of flexible and disordered protein domains remain a significant experimental challenge. Recently, atomic force microscopy (AFM) has emerged as an important complementary tool in biophysics and is well suited for studying membrane protein dynamics in near‐native conditions (i.e., in a native lipid environment, at physiologically relevant temperature and ionic strength). We have studied purified SecYEG that was reconstituted into liposomes via AFM. After confirming activity, changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude and were attributed to the aforementioned loops of SecY. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. Finally, we have imaged SecA engaged on SecYEG and related the structural states observed to the activity of the translocase. This work provides a novel and near‐native vista of central components of the general secretory system.

Grant Funding Source: This work was supported by the National Science Foundation CAREER Award 1054832 (to G. M. K.)
The role of multicopper ferroxidases in mammalian iron homeostasis (995.2)
Authors: Fuqua Brie , Lu Yan , Darshan Deepak , Frazer David , Wilkins Sarah , Page Kathryn , Vulpe Chris , Anderson Gregory
Published: June 2021

Introduction: Multicopper ferroxidases (MCFs) are proteins important for intestinal iron absorption and iron recycling. MCFs are hypothesized to oxidize ferrous iron from the iron exporter Ferroportin so that the iron can bind to circulating Transferrin. There are three known MCFs expressed in vertebrates: Ceruloplasmin (CP), Hephaestin (HP), and the recently discovered Zyklopen (ZP). In order to determine the individual and overlapping roles of the MCFs, we generated mouse models with single, double, and triple MCF knockout. Methods: We used the cre‐lox system to create HP and ZP knockouts and then crossed these mice with each other and with CP knockout mice to generate double and triple knockouts. The general phenotype, hematology, tissue iron levels, and the absorption and distribution of radiolabeled iron were then studied. Results: Mice lacking HP and/or CP exhibited clear defects in iron metabolism, with double knockout leading to a severe anemia and fatality at 23‐30 weeks of age. Mice with knockout of ZP appeared grossly normal except for hair abnormalities including curly whiskers. The phenotypes of double knockout mice lacking ZP and CP or HP were not grossly different than what would be expected from the simple combination of the single knockout phenotypes. Triple MCF knockout mice were viable and able to absorb dietary iron but were severely anemic. Conclusions: Our findings indicate that MCFs are not absolutely required for iron absorption and survival into adulthood. The severity of the phenotype of HP CP double knockout mice indicates that these proteins are particularly important in iron metabolism, while the phenotype of the ZP knockout mice suggests a role for ZP in copper metabolism.
Intestinal divalent metal‐ion transporter‐1 is critical for intestinal iron absorption but not that of copper (996.3)
Authors: Shawki Ali , Anthony Sarah , Engevik Melinda , Niespodzany Eric , Worrell Roger , Mackenzie Bryan
Published: June 2021

Divalent metal‐ion transporter‐1 (DMT1) is a widely expressed iron transporter serving iron absorption and erythroid iron uptake. DMT1 can also transport certain other transition metal ions; however, questions have arisen as to the role that DMT1 plays in the absorption of copper. We tested the hypothesis that intestinal DMT1 is essential for the absorption of Fe and Cu by examining (1) hematological variables and Fe status, (2) expression of Fe‐related genes (by using RT‐qPCR), and (3) radiotracer metal absorption in a mouse model lacking intestinal DMT1 (i.e. DMT1int/int, generated by crossing floxed DMT1 and villin‐Cre transgenic lines). The DMT1int/int mouse (at age ≍ 120 d) exhibited a profound hypochromic–microcytic anemia (hemoglobin concentration was 1.8 ± SD 0.5 g/dL in DMT1int/int mice cf. 14.6 ± 0.7 g/dL in wildtype, n = 7–9), and severely depleted serum Fe and liver nonheme Fe stores. mRNA expression of Cybrd1 (ferrireductase) was increased to (30 ± 3)‐fold (mean ± propagated SE, n = 3) in enterocytes of DMT1int/int mice cf. wildtype mice and that of the basolateral Fe exporter ferroportin, (2.7 ± 1.0)‐fold. Liver Hamp1 (hepcidin) expression was depressed by 97% ± 48%. Expression of the apical Cu transporter Ctr1 was increased to (2 ± 1)‐fold in enterocytes of DMT1int/int cf. wildtype mice, consistent with other models of iron deficiency. Parenteral Fe injection corrected the hematological and serum‐Fe variables, Fe stores, and gene expression levels in the DMT1int/int mouse. We measured the absorption of 59Fe and 64Cu in mice fed by intragastric gavage. Loss of intestinal DMT1 decreased the 0–4‐hour appearance of 59Fe in blood by 88% ± 24% cf. wildtype. Enterocyte 59Fe content at 4 h was 93% ± 42% lower in DMT1int/int cf. wildtype mice, indicating that the defect was at the level of apical uptake. Intestinal handling of 64Cu in the DMT1int/int mouse was no different from wildtype (P = 0.26). We conclude that DMT1 is critical for intestinal absorption of Fe but not Cu.

Grant Funding Source: Supported by PHS Grant DK080047
Identification of accessory proteins necessary for epithelial sodium channel function (997.8)
Authors: Sterling Beau , Booth Rachell
Published: June 2021

The epithelial sodium channel (ENaC) is a selective membrane ion‐channel responsible for the preservation of proper sodium concentration to maintain homeostasis of blood and epithelial fluids. Protein‐protein interactions are important for the regulation, assembly, and membrane insertion and/or retrieval of ENaC. Other proteins necessary for ENaC maturation and function may lead to increased or decreased ENaC activity. Strains from the Saccharomyces cerevisiae Deletion Library were selected on the basis that the deleted gene in that strain may likely interact with ENaC during formation or otherwise regulate its function after integration within the cell membrane. The αENaC gene was transformed into yeast and selected colonies were replica plated using a serial dilution pronging assay. Growth of the yeast expressing αENaC were altered relative to the control strains. Two deletion strains exhibited a significant difference in growth as compared to controls. These strains lacked a CUE1 (elevated growth) and a PEP12 (almost no growth) when expressing αENaC. The CUE1 gene codes for an ubiquitin‐binding endoplasmic reticulum (ER) membrane protein that recruits the ubiquitin‐conjugating enzyme Ubc7p to the ER where it functions in protein degradation. The deleted carboxyPEPtidase Y‐deficient protein, coded by PEP12, is a target membrane receptor (t‐SNARE) for vesicular intermediates that control entry of biosynthetic, endocytic, and retrograde trafficking.

Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmen.tal Grant AI‐0045
Lactosylceramide promotes hypertrophy through ROS generation and activation of ERK1/2 in cardiomyoctes (999.1)
Authors: Mishra Sumita , Chatterjee Subroto
Published: June 2021

Glycosphingolipids (GSLs) are integral components of the surface of essentially all types of cells and tissues including heart. Lactosylceramide (LacCer), a member of the neutral GSL family is synthesized from Glucosylceramide (GlcCer) by Lactosylceramide synthase, plays a pivotal role in the biosynthesis of complex GSLs and imparts several critical phenotypes. However, the role of GSLs in cardiac system is not known and physiological and pathophysiological consequences of myocardial sphingolipid species and their regional differences in myocardium remain obscure. We have determined the efficacy of a wide variety of GSL's to affect cardiac hypertrophy using cultured rat cardiomyocytes e.g. H9C2 cells and cultured primary neonatal rat cardiomyocytes. We employed multiple criteria to assess hypertrophy in our studies. These were: a) measurement of [3H] Leucine incorporation into protein, b) measurement of cell size and morphology and c) Real time quantitative mRNA expression assay for atrial natriuretic protein (ANP) and brain natriuretic peptide (BNP). Subsequently, mechanistic studies were performed to measure the involvement of various signaling transduction pathways.

Our in vitro studies using cardiomyocytes reveal that LacCer dose‐dependently activated the increase in cell surface area, cell volume, protein content and expression of BNP/ANP mRNA. A comparative study using other GSL's did not induce hypertrophy. LacCer induced hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. LacCer also activated MAP kinases implicated in the cardiac hypertrophy signaling pathways. mRNA levels for c‐Jun and c‐Fos were increased by LacCer. Increases in c‐jun and c‐fos gene expression due to LacCer were abolished by inhibition of PKC as well as inhibition of extracellular signal‐regulated kinases (ERK) 1/2. We report here for the first time that LacCer induces hypertrophy in cardiomyocytes.
Channeling Your Inner Mutations (LB104)
Authors: Brinegar Kim , Davis Zachery , Davidson Katie , Fournier Ronald , Hoover Breeanna , Macias Nicholas , Maldonado Vivian , Galea‐Martinez Sandra , Grootenhuis Peter
Published: June 2021

Approximately 70,000 people worldwide have the genetic disease cystic fibrosis (CF). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The protein consists of two membrane spanning domains (MSD1,2), two nucleotide binding domains (NBD1,2), and a regulatory domain (R). The domains ultimately form a chloride channel. When mutant CFTR is not present at the cell surface or is dysfunctional, altered chloride levels lead to the formation of sticky mucus in the lungs, which causes chronic infection and inflammation, and ultimately death. Although there are over 1,900 CFTR mutations known, the most common is the deletion of phenylalanine‐508(F508del), which leads to the misfolding of CFTR and reduced the amount of CFTR at the cell surface. A rare mutation is the G551D mutation, which leads to defective gating of the channel. By targeting two small molecule agents, researchers have made advances in treating the underlying cause of CF. The El Capitan High School SMART (Students Modeling a Research Topic) Team used 3D printing technology to create a model of the CFTR domains based on the crystal structure of related proteins.

Grant Funding Source: Supported by grants from NIH‐CTSA and NIH‐SEPA.
The Role of Yeast Urea Amidolyase in Patients with Suppressed Immune Systems (LB105)
Authors: Cavins K. , Alphin D. , Braatz A. , Deleon‐Camacho F. , Emkay S. , Flees H. , Franitza A. , Gerwig A. , Groth E. , Granlund L. , Idell A. , Klug L. , Nakhla V. , Osberg Z. , Paniagua P. , Wallner J. , Rochon D. , Fangmann J. , St. Maurice Martin
Published: June 2021

According to Rice University, 70% of people are affected by the infectious fungus Candida albicans. The immune system uses T and B cells to stop pathogens. People with suppressed immune systems, such as children with transplants, AIDs or cancer patients, lack functional T and B cells, so they rely on macrophages to destroy Candida. Candida can kill and exit macrophages due to an enzyme: urea amidolyase (UAL). While in the macrophage, Candida goes through a morphological switch from a sphere to a structure with hyphae due to an environment change. UAL converts urea to ammonia and CO2, creating an environment for hyphae to form, bursting the macrophage. The Greenfield SMART (Students Modeling A Research Topic) Team used 3D printing technology to model the four domains of UAL. The biotin carboxylase (BC) domain uses energy from ATP cleavage to attach CO2 to the swinging arm portion, or biotin carboxyl carrier protein (BCCP) domain. The BCCP domain swings across UAL, attaching CO2 to urea forming allophanate in the carboxyl transferase (CT) domain. Allophanate moves to the allophanate hydrolase (AH) domain, which hydrolyzes the allophanate into CO2 and ammonia. Increases in CO2 and ammonia cause hyphae to form, destroying macrophages and allowing Candida to spread. Since humans lack UAL, researchers could block UAL’s active sites to prevent Candida’s macrophage‐killing shape change, thus preventing systemic candidiasis without damaging human cells.

Grant Funding Source: Supported by a grant from NIH‐CTSA.
Efficient Replication of Herpes Simplex Virus Type 1 Involves DNA Damage Pathways (LB122)
Authors: Botting Carolyn , Lu Xu , Triezenberg Steven
Published: June 2021

Herpes simplex virus type 1 (HSV‐1) is a predominant cause of cold sores and is prevalent in 90% of the world’s population. While examining chromatin changes during HSV‐1 infection, we observed histone H2AX phosphorylation, a marker of DNA damage response activation. Since ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3 related (ATR) kinases are responsible for H2AX phosphorylation, we hypothesized that these kinases are involved in HSV‐1 replication. This hypothesis was supported by significant reduction in HSV‐1 replication upon addition of CGK733, an ATM and ATR inhibitor. Small molecule inhibitors specifically targeting either ATM or ATR affected replication only when used in combination, but not to the same extent as that of CGK733. Furthermore, shRNA and siRNAs targeting ATM or ATR caused only a modest reduction in HSV‐1 replication. Together these data suggest a redundant mechanism in which the ATM and ATR pathways may be used interchangeably to promote HSV‐1 replication. Thus new treatment for HSV‐1 could involve simultaneous inhibition of both these pathways.
Investigating cross‐linked peptides in bovine aortic lysyl oxidase (LB129)
Authors: Peng Kai , Greenaway Frederick
Published: June 2021

Lysyl oxidase (LOX) isolated from bovine aorta contains five disulfide bonds and the lysyl tyrosyl quinone (LTQ) cross‐link. In attempting to locate the inhibitor‐modified amino acid residues resulting from addition of various suicide inhibitors, which may be in cross‐linked regions of the protein, we have been optimizing procedures for mass spectrometric identification of cross‐linked peptides produced during proteolysis of the enzyme. To covert the reactive quinone group of the LTQ to an invariant structure, LOX was reacted with phenylhydrazine before proteolysis. Recombinant human and purified bovine lysyl oxidase were first subjected to various proteolyses and mass spectrometry acquisition methods to determine optimal experimental conditions. Proteolytic procedures must produce peptides of manageable size and with termini that can be at least partially sequenced; chymotrypsin proved to be more suitable than trypsin. Optimizing conditions significantly increased the sequence coverage of LOX from 60% to 95%. Targeted MS/MS was used to identify cross‐link containing peptides using calculated m/z values and by utilizing the software packages xQuest/xProphet (ETH Zürich) and pLink (CAS) to find the cross‐link containing peptides.

Grant Funding Source: Supported in part by Nuclea Biotechnologies
Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 (LB130)
Authors: Chakraborty Soumyadeep , Goyal Arun
Published: June 2021

Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 Soumyadeep Chakraborty, Arun Goyal, Indian Institute of Technology, Guwahati, Assam, India A protein sequence with GeneBank accession number ABN53381.1 from Clostridium thermocellum ATCC 27405 belonging to family 1 polysacchaide lyase of CAZy database was scavenged out for this study. This sequence consisted of 352 aa N‐terminal catalytic domain (PL1B) and a 123 aa family 35 carbohydrate binding domain designated as (CBM35) at the C‐terminal. The catalytic domain PL1B was cloned in pET28a expression vector and expressed in E.coli as a soluble protein of molecular size 40 kDa. PL1B was active at alkaline pH on pectic substrates. Optimization of enzymatic reaction conditions revealed that PL1B shows 100% (3005 U/mg) activity at 50ºC in 50 mM Tris‐HCl buffer, pH 9.8. Highest activity was achieved on reaction with non‐methylated polygalacturonic acid (PGA) and almost 75% of the total activity was found with methylated citrus pectin. As this enzyme showed highest activity on non‐methylated PGA hence it was named as pectate lyase. The cleavage mechanism of PL1B towards pectic substrates was determined after analysis of the oligosaccharide produced. The reaction was setup with PGA and citrus pectin separately under optimized conditions of pH and temperature and samples was collected at different time interval at 0 min up to 60 min. Analysis of the reaction products by thin layer chromatography (TLC) revealed that unsaturated di and tri‐galacturonate was produced after enzymatic cleavage together with other oligosaccharides. This finding elucidated that PL1B cleaves within the polysaccharide chain and hence it is an endo cleaving enzyme; thus PL1B can be named as endo‐pectate lyase. Enzymatic degradation products were further analyzed by HPEAC and mass spectrometry.
Identification of H. volcanii Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzymatic Reactions in an Archaeal Alternate Mevalonate Pathway (LB139)
Authors: Miziorko Henry , VanNice John , Skaff D , Keightley Andrew , Addo James , Wyckoff Gerald
Published: June 2021

To identify the enzymes responsible for metabolism of mevalonate 5‐phosphate to isopentenyl diphosphate in H. volcanii, two open reading frames (HVO_2762; HVO_1412) were selected for expression and characterization. Characterization of these proteins indicates that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate. The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate 5‐phosphate, a reaction proposed but never demonstrated by direct experimental proof. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6‐fluoromevalonate monophosphate but negligible inhibition by 6‐fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to the best of our knowledge, the first identification and characterization of an archaeal phosphomevalonate decarboxylase.
MicroRNAs in planarian regeneration (LB14)
Authors: Lin Yu‐Chi , Chen Fang‐Yi , Chuang Chao‐Yi , Chu Chia‐Ying
Published: June 2021

Planarian, with the robust regeneration ability, has been used as a model organism for stem cell research in recent years. Regeneration in planarian is mediated by adult stem cells, called neoblasts, which can proliferate and differentiate into almost all cell types for replacing the whole body. MicroRNAs are 20‐24nt small non‐coding RNAs that are associated with Argonaut proteins and form miRNA‐induced silencing complex (miRISC) to regulate gene expression at post‐transcriptional level. Our preliminary study showed that a certain population of miRNAs are highly expressed in regenerating tissues, implicates the potential important roles of miRNAs in neoblasts during regeneration. Interestingly, depeletion of planarian Argonaute 2 (DjAgo2) results in a dramatic effect of head degeneration, suggesting that miRNAs are also required for planarian homeostasis. In this study, we further examined the roles of specific miRNAs in planarian. We developed a method to specifically block the function of miRNA. Successful inhibition of several candidate miRNAs results in a degeneration phenotype in planarian treated with miRNA inhibitors. This result proved our hypothesis that miRNAs indeed participate in self‐renewal of stem cells and the differentiation process in planarian.
Hepatic carbohydrate and lipid metabolism are altered in rats fed creatine‐supplemented diets (LB151)
Authors: da Silva Robin , Kelly Karen , Jacobs Rene
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of liver damage. Insulin resistance often accompanies NAFLD and these phenomena are hallmarks of the metabolic syndrome. Recently, we reported that creatine supplementation prevents hepatic steatosis, lipid peroxidation and insulin resistance in rats fed a high‐fat diet. In order to examine potential mechanisms underlying these observations, we used McArdle RH‐7777 rat hepatoma cells treated with oleic acid as a model of hepatocyte lipid accumulation. We found that cells cultures with creatine had a dose dependent reduction in cellular TG accumulation. Using radiolabeled tracers we have demonstrated that incubation of hepatoma cells with creatine increases fatty acid oxidation and decreases both fatty acid and TG synthesis. In‐line with increased fatty acid oxidation, analysis of mRNA from hepatoma cells treated with creatine had increased expression of PPARα and its targets CPT1a and LCAD. We have also found that creatine treated hepatoma cells have increased expression of the PEPCK and pyruvate kinase. Our preliminary data suggests that rats fed a creatine‐supplemented high‐fat diet have significantly improved glucose tolerance compared to high‐fat diet fed control animals. Together these data suggest that dietary creatine influences carbohydrate metabolism as well as lipid metabolism.

This research is funded by the CIHR

Grant Funding Source: Supported by CIHR
Mitochondrial integrity is impaired in MELAS patients (LB164)
Authors: Joseph Rosette , Green Alexander , Cameron Jessie , Hood David
Published: June 2021

Mitochondrial diseases are disorders that can be caused by either mitochondrial or nuclear DNA mutations. One of the most prominently studied mitochondrial diseases is mitochondrial myopathy, encephalopathy, lactic acidosis and stroke‐like episodes (MELAS), caused by a mutation in the tRNA‐leucine gene within mtDNA. While the symptoms of this disease have been studied, the molecular manifestations of this mutation remain to be clearly understood. Since alterations in organelle function underlie the pathophysiology of various mitochondrial diseases, we explored mitochondrial morphology, cytochrome c oxidase activity, reactive oxygen species (ROS) production and mitochondrial membrane potential in patient (n=3) and control fibroblast cell lines (n=3). Cells transfected with Mito ds‐Red and visualized using confocal microscopy revealed an increase in mitochondrial fragmentation in cells from MELAS patients, compared to control, coincident with reduced cytochrome c oxidase activity, higher ROS production and a greater membrane potential. The increase in organelle fragmentation observed in MELAS cells led us to investigate the proteins which regulate mitochondrial fission (Fis1 and Drp1) and fusion (Opa1). Our data suggest that the increase in organelle fragmentation was due to a tendency for a higher expression of Fis1 in MELAS cells, suggesting that mtDNA mutations have consequences for the expression of nuclear‐encoded fission proteins, resulting in altered organelle morphology. Future work will investigate the use of fission protein inhibitors to ameliorate mitochondrial function in MELAS cells.

Grant Funding Source: Supported by Mitacs and Panacea Global
Characterization of mitochondrial Lace1 ATPase (LB168)
Authors: Stiburek Lukas , Cesnekova Jana , Houstek Josef , Zeman Jiri
Published: June 2021

Lace1 (Lactation elevated 1) is a human homolog of yeast Afg1 (ATPase family gene 1) ATPase with sequence identity ranging from 41,5 % H. sapiens vs. S. cerevisiae to 88,9 % H. sapiens vs. M. musculus. The protein consists of ATP/GTP binding P‐loop motif and a common five domain structure. The aim of our work was to perform detailed cell biological characterization of human Lace1 utilizing stable shRNA‐based RNA interference approach, proteomics and expression analyses using human embryonic kidney cell line (HEK293). We found that human Lace1 is a mitochondrially localized integral membrane protein with an apparent molecular weight of 50 kDa. The prepared stable shRNA LACE1 knockdown HEK293 cell line showed markedly reduced Lace1 protein levels (<10% of controls). SDS‐PAGE western blotting demonstrated markedly elevated levels of subunit SDHA of respiratory complex II, Cox2, Cox3, Cox4, Cox5a and Cox6aL subunits of respiratory complex IV, and ATP d subunit of complex V. On the other hand, the amount of the antiapoptotic factor Bcl‐2 was found to be decreased in these cells. Furthermore, slightly elevated levels of the tumor suppressor p53 were found in LACE1 knockdown cells. Quantitative 2D‐PAGE analysis coupled to mass spectrometric identification (MS) showed elevated accumulation of chaperonine 10 and ATPase F1‐alpha. Our work have thus far identified Lace1 as a mitochondrial factor playing a role in protein turnover of subunits of the oxidative phosphorylation system, possibly confirming its predicted function as an adaptor protein of mitochondrial proteases. Future experiments will involve short term downregulation of Lace1 using Stealth siRNA approach as well as co‐immunoprecipitation analyses coupled to MS identification with P‐loop mutant Lace1 protein.

Grant Funding Source: Grant Agency of the Czech Republic Project GACR 13‐072235 and institutional projects UNCE 204011 and RVOVFN6465.
Profiling metabolic and bioenergetic properties of 3D tumor microtissues (LB171)
Authors: Rogers George , Dranka Brian , Swain Pamela , Divakaruni Ajit , Neilson Andy , Murphy Anne , Ferrick David
Published: June 2021

Cellular metabolism and bioenergetics processes are increasingly recognized as critical regulators in the pathogenesis of cancers. Metabolic activity and bioenergetic processes are often characterized using typical 2 dimensional (2D) adherent cell culture systems; however, this approach lacks the 3 dimensional (3D) context and heterogeneity of tumor tissue in vivo, which may have significant effects on metabolic and bioenergetic behavior. A novel and more physiologically relevant model is the use of microtissues (also known as spheroids). Metabolic activity and bioenergetic function were accessed in individual cancer cell line microtissues using a newly designed 96‐well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer. Mitochondrial and glycolytic functions were determined simultaneously using the Cell Mito Stress and Glycolytic Stress Tests, respectively. Several parameters, including: seeding density, microtissue size, and substrate identity and availability were investigated. Further, the potential of the microtissues to switch bioenergetic pathways was determined by stimulating maximal respiration (OCR) and maximal extracellular acidification rate (ECAR). Comparisons of microtissues to typical 2D adherent cell cultures were also performed. Initial comparative data are consistent with a shift from the highly proliferative nature of 2D cultures to a physiological 3D model where growth and metabolic properties of the cell are balanced. Technical aspects of this assay system and implications of this work for future studies in cancer biology will be discussed.
Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly (LB185)
Authors: Al‐Hadid Qais , Roy Kevin , Chanfreau Guillaume , Clarke Steven
Published: June 2021

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine‐243, a residue that contacts the 25S rRNA near the P‐site. Rpl3p methylation is dependent upon Hpm1p, a candidate seven beta strand methyltransferase. In this study, we characterized Hpm1p activity in vitro and in vivo. Amino acid analysis of cell lysates and subcellular fractions reveals that Hpm1p is responsible for all detectable histidine protein methylation. The modification is found in a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nuclei‐containing organelle fraction, but was not detected in proteins of the ribosome‐free cytosol fraction. In vitro assays demonstrate that Hpm1p is a methyltransferase that catalyzes the modification of ribosome‐associated but not free Rpl3p, suggesting that its activity depends on interactions of Rpl3p with other ribosomal components. hpm1 null mutant cells are defective in early rRNA processing resulting in a deficiency of 60S subunits and decreased translational activity in minimal media. Finally, cells lacking Hpm1p are resistant to cycloheximide, indicative of ribosome structure abnormalities. We propose that Hpm1p plays a role in orchestrating the assembly of biogenesis factors during early ribosome assembly.

Grant Funding Source: NIH
Identification of ICER’s ubiquitination sites to distinguish its Novel Ubiquitin Ligase (LB186)
Authors: Woldeamanuel Fanaye , Molina Carlos
Published: June 2021

Inducible cAMP Early Repressor (ICER) is a dominant negative transcriptional repressor that expresses tumor suppressor activity. In cancer cells, ICER is abnormally expressed and becomes modified by the posttranslational modification ubiquitination. Consequentially, ICER is degraded or mislocalized from the nucleus to the cytosol where it is non‐ functional. When exogenous ICER is introduced into cancer cells, it inhibits the transformed phenotype of cancer cells. The goal of this project is to identify the ubiquitinated residues of ICER to ultimately rescue this protein from degradation and cytosolic localization. Since lysine residues are sites for ubiquitination, mutant forms of each residue were constructed using site‐directed mutagenesis. All lysine residues were substituted to arginine to create an ICER molecule without lysines. Another 10 independent mutants were generated where all but one specific lysine was substituted by arginine. All clones were subcloned in a mammalian expression vector. By using these altered forms of ICER, we have found that ICER is ubiquitinated in several lysines. This data suggested alternative modes of regulating ICER degradation and subcellular localization. The long‐term goal of this project is to identify ways of inhibiting ICER degradation in cancer cells as a new mode for cancer treatment.
Modular organization and conformational variability of the yeast SAGA complex (LB187)
Authors: Setiaputra Dheva , Ross James , Yip Calvin
Published: June 2021

Spt‐Ada‐Gcn5‐Acetyltransferase (SAGA) is a conserved histone acetyltransferase complex that regulates transcription through acetylation and deubiquitination of nucleosomal histones. The 19 subunits constituting SAGA are thought to organize into four modules. To further elucidate the relationship between these modules, we developed an improved purification method that enhances the stability of SAGA and used single‐particle electron microscopy to examine the overall architecture and organization of this complex. Our two‐ and three‐dimensional analyses revealed that SAGA is flexible and adopts three major conformations involving large‐scale rearrangements of its subunits. Subunit deletion and labeling showed that the acetyltransferase module is located in the most mobile region of the complex, and that the loss of the deubiquitination module reduces SAGA’s ability to adopt one specific conformation. These results lead to a working model in which the different modules of SAGA coordinate to mediate structural remodeling, which in turn accommodates binding of various substrates and transcriptional activators.
Phosphorylation regulates myo‐inositol 3‐phosphate synthase: A novel regulatory mechanism of inositol biosynthesis (LB190)
Authors: Deranieh Rania , He Quan , Caruso Joseph , Greenberg Miriam
Published: June 2021

Myo‐inositol phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate (VPA), suggesting that the enzyme is post‐translationally regulated. Using 32Pi ‐ labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation‐deficient and phosphomimetic site‐mutants indicated that the three conserved sites in yeast (S184, S296, and S374) and human (S177, S279, and S357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phospho‐deficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374 caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to VPA. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS

Grant Funding Source: Supported by National Institutes of Health Grant DK 081367, and Wayne State University Graduate Research Fellowship and Graduate Research Enhancement Funds
Histone H3 lysine 4 methyltransferase subunits regulate cytokinesis (LB191)
Authors: Bailey Jeffrey , Fields Alexander , Cheng Kaijian , Lee Albert , Wagenaar Eric , Lagrois Remy , Schmidt Bailey , Xia Bin , Ma Dzwokai
Published: June 2021

Cytokinesis, the physical separation of two dividing cells, occurs at the midbody and affects the growth and development of all organisms. As the final step of cytokinesis, abscission requires membrane fission and cytoskeletal rearrangement at a specific midbody region called the secondary ingression. Here, we report that histone H3 lysine 4 methytransferase (H3K4MT) subunits reside at the midbody. Knockdown of individual H3K4MT subunits increases the incidence of multinucleated cells, slows cell proliferation, and impairs cytokinesis at the abscission step. Further investigation shows that the abscission delay is due to slower formation of secondary ingressions in the knockdown cells. Importantly, the midbody microtubules in H3K4MT knockdown cells display enhanced resistance to depolymerization by nocodazole and the abscission defect in these cells can be partially rescued by drug‐induced microtubule disassembly. We propose that a midbody‐localized H3K4MT complex or subcomplex promotes abscission, at least partially, by facilitating midbody microtubule disassembly.

Grant Funding Source: 5R21‐MH086853‐02
Increased rate of endocytosis in calreticulin knockout MEF cells is mediated via Caevolin‐1 overexpression (LB195)
Authors: Viswanathan Divya , Pillai Dhanya , Massaeli Hamid , Mesaeli Nasrin
Published: June 2021

Calreticulin (CRT) is a highly expressed protein in mammalian cells, with both calcium binding and chaperone activity. As an endoplasmic reticulum chaperone, CRT is involved in quality control process during the folding and maturation of proteins. Previous reports illustrated that loss of CRT function (crt‐/‐) results in onset of ER stress, elevated ubiquitin‐proteasome activity, and increased resistance to apoptosis. We recently observed a significant increase in connexin 43 expression accompanied with a significant decrease in its function and accumulation of connexin 43 in intracellular vesicles. Thus we hypothesized that loss of CRT function increases rate of endocytosis. To test this hypothesis we examined rate of endocytosis using Wheat Germ Agglutining (WGA). Our data showed that in crt‐/‐ WGA was completely endocytosed within 30 minute incubation while in wild‐type cells WGA was localized both on cell membrane and intracellular vesicles at the same time. Furthermore, inhibiting proteasome with MG132 reduced endocytosis rate in crt‐/‐ cells. Western blot analysis illustrated a significant increase in caveloin‐1 protein level in crt‐/‐ cells. Quantitative real time PCR showed 3 fold increase in expression of caveolin‐1 in crt‐/‐ compared to wt cells. Furthermore, ubiquitin pull down experiment showed significantly higher caveolin‐1 ubiquitination in crt‐/‐ compared to wt cells. Our data show for the first time involvement of CRT in endocytotic pathways. Furthermore, we showed that knock out of CRT gene results in enhanced endocytosis that is dependent on the ubiquitin‐proteasome activity.

Grant Funding Source: Qatar National Research Foundation