bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis of inter‐domain electron transfer in human Ncb5or
Authors: Deng Bin , Parthasarathy Sudharsan , Wang WenFang , Lovell Scott W. , Benson David R. , Zhu Hao
Published: June 2021

NADH cytochrome b5 oxidoreductase (Ncb5or) is found in all animals and contains three domains: cytochrome b5 (b5), CHORD‐SGT1 (CS) and b5 reductase (b5R). The two redox domains are homologous to microsomal b5 (Cyb5A) and b5R (Cyb5R3), the two known electron donors of stearoyl‐CoA desaturase in vitro. To elucidate the structural basis of inter‐domain electron transfer in human Ncb5or, we generated individual b5 and b5R domains for structural and kinetic studies. The crystal structure of Ncb5or‐b5 was solved by X‐ray diffraction with a resolution of 1.95 Å. There are fewer surface charged residues in Ncb5or‐b5 than Cyb5A, suggesting a weaker electrostatic interaction in b5‐b5R than Cyb5A‐Cyb5R3. This was confirmed in our measurements of electron transfer in b5‐b5R, Cyb5‐Cyb5R3 and mismatched pairs. The linkage through CS likely obviates the need for strong interactions between b5 and b5R in Ncb5or. Ncb5or‐b5 has two helix‐loop‐helix motifs surrounding heme, but differs from Cyb5A in that helix 4 is canted at 45º and shorter. The heme‐ligating His112 residue between helix 3 and 4 is displaced due to one‐residue deletion (before) and insertion (after). Trp114 in helix 4 of Ncb5or‐b5 is a well conserved surface residue potentially involved in inter‐domain recognition or electron transfer. We thus conclude that the Ncb5or‐b5 core has a conserved b5 scaffold with unique features. (Supported by NIH)
Characterization of a dimeric Arginine kinase in the protozoan, Phytophthora sojae, suggests an early origin of Phosphagen kinase dimers
Authors: Palmer Allyson Kaitlin , Fraga Dean , Snider Mark , Edmiston Paul
Published: June 2021

The Phosphagen kinase (PK) enzyme family plays a critical role in energy homeostasis by catalyzing the reversible transfer of a high‐energy phosphoryl group from ATP to a guanadino‐containing acceptor molecule. Creatine kinase (CK), found in vertebrates and some invertebrates, exists as a dimer. Arginine kinase (AK), found in invertebrates, protozoa, and some bacteria, usually exists as a monomer, but several dimeric AKs have been characterized. The evolutionary relationship between these two forms of AK and the CK is unclear. Dimeric AKs described previously in invertebrates were proposed to have derived from CK, however multiple separate occurrences of this ‘back evolution’ make this scenario problematic. Using sequence motifs known through previous work to be important for dimerization in PKs, we identified an AK in a protozoan, the oomycete Phytophthora sojae, that we predicted would be dimeric. This putative dimeric AK has been cloned and expressed and shown to exhibit AK activity. More importantly, preliminary size‐exclusion chromatography data indicate that it is likely a dimeric enzyme. To expand this characterization, isothermal titration calorimetry will be used to determine whether the enzyme exhibits subunit cooperativity. These data imply that dimeric AKs were present much earlier than previously thought and suggest a different evolutionary history for PK enzymes than previously proposed.
Metazoan mitochondrial alanyl‐tRNA synthetases – varying roles for a C‐terminal domain
Authors: Chihade Joseph , Anderson David , Borchert Karen , Brown Julia , Mork A. Jolene , Natala Nakita , Zhu Yirong
Published: June 2021

Metazoan mitochondrial tRNAs are unusual.

Some differences may affect tRNA identity, and are likely to result in compensatory changes in the corresponding aminoacyl‐tRNA synthetase (aaRS). We have focused our attention on two mitochondrial AlaRSs, from humans and C. elegans.

Human mitochondrial (hm) tRNAAla lacks a G3:U70 wobble pair, a conserved identity element in non‐mitochondrial alanine tRNAs. Aminoacylation by hmAlaRS is insensitive to changes in the tRNA acceptor stem, but sensitive to changes in the variable loop‐anticodon stem. A C‐terminal domain, dispensable in non‐mitochondrial systems, is required for aminoacylation by hmAlaRS.

In C. elegans mitochondrial (cem) tRNAAla a six‐nucleotide sequence replaces the usual T stem‐loop and variable loop. Unlike paralogs, cemAlaRS aminoacylates microhelix substrates as efficiently as full‐length tRNAs, suggesting that relevant contacts are only with the acceptor stem of the tRNA. Interestingly, cemAlaRS lacks a C‐terminal domain conserved in non‐mitochondrial systems.
Freshwater mollusc thiolase cloning, expression and structural analysis
Authors: Dyer James H. , Prezant Robert
Published: June 2021

A considerable gap remains in the knowledge of lipid metabolism and lipid biochemistry in molluscs. Many species of freshwater molluscs exist in North America. Among these are the common gastropods Helisoma trivolvis, Physa acuta, and Goniobasis virginica. Recent advances, including high‐resolution x‐ray crystal structures, in structural analysis of acetoacetyl CoA thiolase from various species has advanced the understanding of the catalytic mechanism of these important enzymes. The purpose of this research is to clone and express thiolase enzymes in various freshwater molluscs and to use other known thiolase structures to study the structure of the mollusc thiolases.
Novel anti‐aging small molecules greatly extend yeast life span by specifically targeting a mechanism underlying the essential role of cellular lipid movement and compartmentalized metabolism in regulating longevity
Authors: Titorenko Vladimir , Goldberg Alexander , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent , Kyryakov Pavlo , Gregg Christopher , Boukh‐Viner Tatiana , Juneau Mylène
Published: June 2021

We use the yeast Saccharomyces cerevisiae as a model to study the mechanisms linking lipid dynamics and longevity. Yeast aging can be slowed down by calorie restriction (CR), a diet that extends life span and delays age‐related disorders in various organisms. We assessed the effect of CR and numerous life‐extending mutations on the spatiotemporal dynamics of the proteomes and lipidomes of organelles involved in lipid metabolism, including the endoplasmic reticulum (ER), peroxisomes and lipid bodies. Our findings revealed a mechanism underlying the essential role of lipid dynamics in regulating longevity. In this mechanism, a calorie‐rich diet suppresses peroxisomal oxidation of free fatty acids (FFA) that originate from neutral lipids synthesized in the ER and deposited within lipid bodies. The resulting accumulation of FFA initiates several negative feedback loops regulating the metabolism of neutral lipids, ultimately leading to the accumulation of diacylglycerol (DAG). The buildup of FFA promotes necrotic cell death, whereas the accumulation of DAG hampers a stress response‐related signal transduction network. We identified 25 novel anti‐aging small molecules that greatly extend yeast longevity by remodelling compartmentalized lipid metabolism and preventing the FFA‐induced necrotic cell death. Our findings revealed two different ways for delaying aging by altering cellular lipid dynamics.
The Role of Interleukin 1 Receptor Associated Kinase 1 (IRAK1) in lipid metabolic alterations associated with endotoxemia
Authors: Chang Samantha Mee , Maitra Urmila , Li Liwu
Published: June 2021

Both inflammatory and metabolic alterations have been established in the pathology of endotoxemia and septic shock, making treatment very difficult. The metabolic alterations associated with endotoxemia contribute to multiple organ failure (MOF) and high mortality rate in septic patients. However, the mechanism behind the shift in metabolism during lipopolysaccharide (LPS) ‐mediated endotoxemia has not been defined. In this study, we examine the role of Interleukin 1 receptor associated kinase 1 (IRAK1), a kinase downstream of the innate LPS receptor Toll‐like receptor 4 (TLR4), in the lipid metabolic alterations associated with LPS. We report a decrease in fatty acid oxidation (FAO) and an increase in plasma levels of free fatty acid (FFA) and triglyceride (TG) in WT but not IRAK1−/− mice injected with a lethal dose of LPS (25mg/kg). Mechanistically, we demonstrate that LPS suppresses FAO by decreasing the expression of mitochondrial β‐oxidation enzymes including carnitine palmitoyl transferase 1□ (CPT1□) and medium chain acyl dehydrogenase (MCAD), as well as pyruvate dehydrogenase kinase 4 (PDK4). Additionally, LPS suppressed the levels of nuclear receptors PPAR□ and PGC‐1□ in tissues from WT but not IRAK1−/− mice after lethal LPS injection. In conclusion, this study establishes that IRAK1 plays a novel role in the lipid metabolic alterations associated with TLR4 stimulation by LPS.

Research funded by the NIH T32 Program, mentorship provided by Dr. Liwu Li, member of the ASBMB.
Phosphatidylethanolamine modification contributes to levuglandin/isoketal induced cytotoxicity
Authors: Davies Sean Stephen , Sullivan C. Blake , Matafonova Elena , Roberts L. Jackson , Amarnath Venkataraman
Published: June 2021

Background

Levuglandins and isoketals are γ‐ketoaldehyde isomers (IK) formed by non‐enzymatic rearrangement of prostaglandin H2 and H2‐isoprostanes, respectively. IK rapidly adduct to proteins and increases in IK protein adduct levels occur in several inflammatory conditions. IK induce cell death in cultured cells, potentially by adducting to cellular proteins. However, IK also adduct to other primary amines including phosphatidylethanolamine (PE) in vitro. Whether IK induces its biological activities by modifying PE has not been investigated.

Objective

Measure levels of IK‐PE in cells and determine whether IK‐ PE mediates the endothelial cell death induced by IsoK.

Results

40% radiolabeled IK added to HEK cells bound to phospholipids and only 8% with proteins. An LC/MS/MS assay using S. chromofuscus phospholipase D to convert IK‐PE to IK‐ethanolamine was used to measure IK‐PE adducts in IK treated endothelial cells (EC). IK‐PE added directly to EC dose‐dependently induced cytotoxicity (LC50 2.2 μM).

Conclusion

PE is a major cellular target of IK, and IK modified PE can mediate IK induced cytotoxicity.
Interdisciplinary Studies of the Multifaceted C2 Domains
Authors: Stahelin Robert V , Yu Hui , Olmstead Deborah , Gunasekaran Christopher
Published: June 2021

The C2 domain (~130 residues) was originally identified as one of two conserved regulatory domains of Ca2+‐dependent protein kinases C. A large number of proteins containing the C2 domain have been identified since, and most of them are involved in signal transduction or membrane trafficking. Early experiments demonstrated C2 domains could bind bulk phospholipids such as phosphatidylserine or phosphatidylcholine with high affinity and specificity, while more recently; C2 domains have been shown to interact with different phosphoinositides and/or sphingolipids in both Ca2+‐dependent and ‐independent manners. C2 domains have also been shown to bind specific proteins, interact with phosphotyrosine, induce membrane curvature changes, participate in intramolecular interactions, and interact with viral proteins. How the multifaceted C2 domains achieve diverse signaling and interaction properties is only beginning to come to fruition. We have begun a comprehensive and rigorous experimental, computational, single molecule, and structural analysis of the mammalian C2 domain family, which will lead to a better understanding of how these properties manipulate the full‐length proteins’ functions.
Quantification of glycerophospholipids in Escherichia coli
Authors: Hopson Marquise L , Garrett Teresa A
Published: June 2021

Phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) are the major glycerophospholipids in Escherichia coli. Each of these lipid classes contains numerous possible molecular species as defined by the total number of carbons and unsaturations in the acyl chains. The goal of this project is to quantify the total amount of each molecular species of PA, PE, PG, and CL in E. coli cells grown under different conditions. Wildtype E. coli was grown at 37°C and the lipids were extracted from cells in the presence of thirteen synthetic standards: three PE, PA, and PG and four CL standards. The samples were analyzed using normal phase liquid chromatography electrospray ionization mass spectrometry. Peak areas for the extracted ion current were recorded for each endogenous glycerophospholipid species and compared to its corresponding standard to determine the nmol of each molecular species of PE, PG, PA, and CL. The amount of each molecular species was summed to calculate the percent composition of PA, PE, PG, and CL in E. coli cells. The observed quantities, PE 79%, PG 19%, PA 1%, and CL 0.2%, are consistent with previous literature. In future experiments, the phospholipid composition in E. coli will be measured under various conditions, such as growing cells in different temperatures and under conditions that may alter acyl chain composition.
Effect of Precursor Structures on the Action of Heparan Sulfate Biosynthetic Enzymes
Authors: Victor Xylophone V , Babu Ponnusamy , Nelsen Emily , Kuberan Balagurunathan
Published: June 2021

Heparan sulfate (HS) plays major role in various physiological and pathological pathways by acting as co‐receptor for a variety of growth factors. The specificity of these diverse functions of HS is attributed to a diverse array of specific sulfation patterns that are present in the HS molecules. The HS biosynthetic enzymes that generate this variety of distinct structures are N‐deacetylase‐N‐sulfotransferases, HS C5‐epimerase, 2‐O‐sulfotransferase, 6‐O‐sulfotransferases and 3‐O‐sulfotransferases. Most, if not all, of these enzymes are expressed as multiple isoforms that have different substrate specificities, and are also differentially expressed in a spatiotemporally controlled manner. In this study, we investigated the effect of various precursor structures on the enzymatic activity of HS biosynthetic enzymes and their isoforms. Our results indicate for the first time that certain modifications in the precursor structures can impact the action of the biosynthetic enzymes to generate distinct structures. Furthermore, these results provide insights in to the GAGOSOME‐mediated generation of combinatorial HS structures that regulate growth factor interactions and subsequent biological action.
Mutations in fibrillin‐1 leading to classical and neonatal Marfan syndrome cause differential protease susceptibilities and protein function
Authors: Hubmacher Dirk , Kirschner Ryan , Iyengar Garud , Bromme Dieter , Bartels Rainer , Reinhardt Dieter P.
Published: June 2021

Fibrillin‐1 constitutes the backbone of extracellular matrix microfibrils, which are crucial for regulating elastic fiber biogenesis and TGF‐beta bioavailability. Mutations in fibrillin‐1 give rise to the Marfan syndrome (MFS) characterized by vascular, skeletal and ocular symptoms. To investigate molecular consequences of mutations causing severe neonatal or the milder classical form of MFS, point mutations from each group were introduced in recombinant human fibrillin‐1 fragments. Proteolytic susceptibility was probed with physiological and non‐physiological proteases. The mutant proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage than those with classic mutations. The cleavage sites were found both in close proximity and distant to the mutations, indicating structural changes and the exposure of cryptic cleavage sites. The group of neonatal mutations more severely affected the ability of fibrillin‐1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. Fibroblasts attached less efficiently to the neonatal as compared to the classical mutant proteins. These results suggest new molecular pathogenetic concepts for MFS. In most cases the neonatal mutations resulted in more severe effects, and the biochemical variability correlates with the clinical variability observed in MFS. The work was funded by the Canadian CIHR.
Biochemical Response And Secretion Of Extracellular Protein And Carbohydrate By Pseudomonas Putida Grown On Fluoranthene And Hydrogen Peroxide
Authors: Igwo‐Ezikpe Miriam Nwanna , Gbenle George Olabode , Ilori Matthew Olusoji , Okpuzor Joy , Osuntoki Adediran Akinniyi
Published: June 2021

The understanding of microbial biochemical responses is paramount in the clean‐up of polluted environment and as such can not be over emphasized. Pseudomonas putida oxidative stress enzymes response and extracellular fluid emulsification activities during the growth of the isolate in mineral salt media containing varying concentrations of fluoranthene and hydrogen peroxide was investigated. Catalase and lipid peroxidation activities increased while there were no significant (P < 0.05) changes in superoxide dismutase and glutathione‐S‐transferase activities. There was also significant (P < 0.05) increase in P. putida biomass and total extracellular protein and carbohydrate content of the growth media compared to controls in the absence of the substrate. Extracellular fluid of the growth media did not exhibit emulsification activities on kerosene, diesel oil, engine oil, hexadecane, dodecane, xylene and hexane. Present study showed that oxidative stress enzymes of the isolate could be induced during degradation of the pollutants cushioning their toxic effects on the microbe with associated secretion of extracellular proteins and carbohydrates which may be involved in solubilization of the substrates.
Secretor phenotype and genotype are novel predictors of severe outcomes in premature infants
Authors: Morrow Ardythe L. , Meinzen‐Derr Jareen , Huang Pengwei , Schibler Kurt R. , Cahill Tanya , Kallapur Suhas G. , Newburg David S. , Tabangin Meredith , Warner Barbara B. , Jiang Xi
Published: June 2021

Background

The secretor gene (FUT2) and its products have important role in mucosal maturation, immunity, and host susceptibility to pathogens. We conducted the first investigation of secretor phenotype and genotype of premature infants in relation to their risk of death, necrotizing enterocolitis (NEC; Bell's stage II and III), and confirmed sepsis.

Methods

Secretor phenotype was measured in infant saliva samples by enzyme immunoassay of H antigen, the primary antigen produced by transferases of secretor gene. The optimal cut‐point in salivary H values was identified and confirmed by computerized algorithms (CART and ROC analysis).

Results

Of the 410 study infants, 14% with low salivary H died, compared to 2% of infants with high salivary H (p<0.001). Modeled with clinical covariates, low H remained significant, and the overall model highly predicted death. Low H antigen was also associated with 10‐fold or greater odds of NEC and sepsis deaths, 3‐fold higher odds of surgical NEC and 2‐fold higher odds of NEC. Similarly, secretor genotype was a significant predictor: 13% of non‐secretors, 5% of heterozygotes, and 2% of secretor dominant infants died (p=0.01).

Conclusions

Lack of secretor antigen predicts adverse outcomes in prematurity. Sponsorship NICHD HD13021, HD059140
The Goldilocks Effect: Identifying the Mechanism by Which O‐GlcNAc Regulates the expression of OGT and O‐GlcNAcase in Response to Changes in Cellular State
Authors: Nimaga Bahawa , Doshi Shivang , Zachara Natasha E
Published: June 2021

Over 500 nuclear, cytoplasmic and mitochondrial proteins are modified and regulated by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc). The addition and removal of O‐GlcNAc to proteins is catalyzed by just two enzymes, the O‐GlcNAc transferase and the O‐GlcNAcase. Previously, it has been shown that when O‐GlcNAc levels are raised exogenously, the cell alters the expression of OGT (down) and O‐GlcNAcase (up) to return O‐GlcNAc levels to normal. We refer to this phenomena as the “goldilocks effect”. The mechanisms by which the cell fine‐tunes the expression of OGT and O‐GlcNAcase are unknown.

In this study we have shown that: 1) When OGT is deleted, the expression and activity of O‐GlcNAcase are reduced. However, the level of O‐GlcNAcase transcript is not affected suggesting that the expression of O‐GlcNAcase in the cell is regulated by protein turnover. 2) In cells with O‐GlcNAc, but deficient in OGT, O‐GlcNAcase expression is maintained, suggesting that the expression of O‐GlcNAcase is dependent on the level of O‐GlcNAc. 3) In cells with exogenously elevated levels of O‐GlcNAc, O‐GlcNAcase expression was elevated. But, when these cells were stressed, a cellular state in which elevated O‐GlcNAc levels are desirable, O‐GlcNAcase over‐expression was ablated. Together these data suggest that the cell can sense both the level of stress and O‐GlcNAc in the cell, and adjust the expression of OGT and O‐GlcNAcase accordingly.
Modulation of sarcoma cell movement and matrix interaction following manipulation of cell surface proteoglycans
Authors: Rossi Silvia , Lacrima Katia , Garusi Elena , Nicolosi Pier Andrea , Affò Silvia , Gulli Mariolina , Gobbi Giuliana , Vitale Marco , Perris Roberto
Published: June 2021

Cell surface proteoglycans are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis formation. Using soft‐tissue sarcoma as model we are trying to understand if different combinations of cell surface PGs can modulate tumour cell interaction with the host microenvironment and metastasis formation; so we are systematically modifying the constitutive patterns of these cells by in vitro gene transduction and RNAi knockdown of individual PGs in different sarcoma cell lines. Overexpression of GPC4, 5 and 6 shows a modulation of the other surface PGs and these new patterns of expression differentially affects the cell migratory and adhesive behaviour on purified ECM molecules and native matrices. RNAi‐mediated knockdown of some SDCs and GPC6 alters cell motility in vitro. Glypicans seem to undergo complex internalisation processes suggesting that endocytic pathways associated with these PGs may control the locomotory machinery. Both syndecans and glypicans seem to influence cell adhesion depending on the matrix substrate. Phospho‐proteomic screenings are in progress. Manipulated cells are assayed for their ability to form tumours and metastasis in mice. Our present findings are starting to delineate correlations between a given repertoire of surface PGs and malignant behavior of sarcoma.
Cleavage of Arrestin3 by Caspases Prevents Apoptosis via Modulation of JNK‐dependent Cell Death Pathway
Authors: Kook Seunghyi , Gurevich Vsevolod , Gurevich Eugenia
Published: June 2021

Ubiquitously expressed arrestin2 and arrestin3 (ARR3) regulate numerous GPCRs and many other signaling proteins, including key players in life‐or‐death decisions, such as Src, ERK, and JNK. Here we show that arrestin3 is specifically cleaved at Asp366, Asp405 and Asp406 by caspases during apoptosis induced by a variety of stimuli in different cell types. Caspase‐generated ARR3(1–366) dose‐dependently increases the survival of etoposide‐treated cells, whereas caspase‐resistant D366/405/406E mutant significantly facilitates apoptosis. We recently showed that all four vertebrate arrestins bind the three kinases in ASK1‐MKK4‐JNK3 cascade, but only arrestin3 facilitates the activation of pro‐apoptotic JNK3. We found that the deletion of ARR3 C‐terminus by caspases does not prevent JNK3 binding, but abolishes its ability to activate JNK3. In contrast, caspase‐resistant D366/405/406E mutant activates JNK3 as effectively as WT ARR3. Thus, caspase‐generated ARR3(1‐366) prevents signaling in ASK1‐MKK4‐JNK3 cascade and protects cells. These findings demonstrate the involvement of ARR3 in the regulation of apoptotic cell death via JNK3 activity.

NIH grants NS065868 (EVG), GM077561, GM081756 (VVG)
Role of Mst3 in oxidative phosphorylation
Authors: Lin Chia‐Ying , Wu Hung‐Yi , Yuan Chiun‐Jye
Published: June 2021

Mst3 (mammalian Ste20‐like serine/threonine protein kinase 3), a key participant in the stress‐ and drug‐induced apoptosis, was recently shown be present not only in cytoplasm but also in the intermembrane space of mitochondria by co‐immunofluorescent staining, scanning electron microscopic imaging and immuno‐blotting after subcellular fractionation. Similar results were also observed in human trophoblast cell line 3A‐sub‐E cells, suggesting that the mitochondrial localization of Mst3 is a general phenomenon. Interestingly, the selective knockdown of Mst3 by RNA interference (RNAi) resulted in a significant reduction of the protein level of respiratory transport chain complexes I and III. Further studies showed that HeLa stable clone with selective knockdown of Mst3, HeLa(siMst3), exhibited an increased dependence of glucose for ATP synthesis. Compared with controls, the survival of HeLa(siMst3) stable clone was much sensitive to the deprivation of glucose in the cultural medium leading to apoptosis. The results indicateMst3 plays an important role not only in cell apoptosis but also in physiological function in cell alive.
Ischemia‐Reperfusion Induces Transcriptional Activation of Brain Natriuretic Peptide Gene via ERK1/2 in H9c2 cells.
Authors: Choe Yun‐jung , Ko Kyoung‐Won , Lee Sun‐Young , Youn Ho‐Joong , Kim Ho‐Shik
Published: June 2021

Brain natriuretic peptide (BNP) is a cardiac hormone released from heart ventricular cells in response to myocyte stretch. Although BNP has recently been shown to be cardioprotective during myocardial ischemia/reperfusion (IR), mechanism of BNP gene expression by IR has not been clarified yet. Here, we show the mechanism underlying IR‐induced BNP gene expression in H9c2 rat cardiomyocytes.

IR induced expression of BNP mRNA and transactivation of BNP promoter in H9c2 cells. Activation of MAP kinase family members such as ERK1/2 and p38 MAPK was triggered by IR. U0126, an inhibitor of MEK, but not SB203580, an inhibitor of p38 MAPK, completely prevented increase of IR‐induced BNP gene expression. In addition, antioxidants such as DMSO and TEMPO diminished induction of BNP and phosphorylation of ERK1/2. To investigate whether increase in free intracellular calcium [Ca2+]i has a role in expression of BNP by IR, calcium chelator BAPTA‐AM was treated before reperfusion. BAPTA‐AM strongly reduced induction of BNP by IR. Moreover, calcium ionophore A23187 increased levels of BNP mRNA and phosphorylation of ERK1/2. Taken together, IR‐mediated BNP gene expression may be due to calcium‐ and ROS‐mediated activation of ERK1/2 in H9c2 cells. This research was supported by MRC from cell death disease research center funded by KOSEF.
Unbiased Functional Annotation of the Human Regulatome
Authors: Mrowka Ralf , Persson Pontus B , Blüthgen Nils
Published: June 2021

Understanding the functional relevance of regulatory elements and their associated genes is a major goal in the postgenomic aera. Recently, a comprehensive catalogue of human regulatory elements based on phylogenetic foot printing analysis has been established. This set of contains more than 50% new motifs (105), which show no similarity to a binding site bound by a known factor. Here, we introduce a new method and analyse large scale genome wide microarray data together with 21921 putative promoters and predict the gene ontology categories of the regulatory elements contained in the catalogue. We are able to map 526 functions to 72 regulatory elements of the catalogue. We show that the new method greatly enhances the functional prediction power in the analysis pipeline. We rigorously validate our new method based on an unbiased “literature wide”‐whole‐Pubmed analysis of known transcription factors as well as based on random background models. We estimate that only 3% of our new predictions are false positive.
Gene expression profile of Staphylococcal Enterotoxin B and Lipopolysaccaride induced human PBMCs
Authors: Bandle Jorge , Hildebrandt Suzanne , Jett Marti , Mendis Chanaka
Published: June 2021

Staphylococcal enterotoxin B (SEB), an exotoxin produced by staphylococcal aureus, is commonly associated with food poisoning and capable of triggering flu‐like symptoms resembling those of endotoxins – such as lipopolysaccharide (LPS) released from Gram‐negative bacterial cell walls. By comparing the host's response to these two toxins on a genetic level, a set of genes was identified to facilitate the differentiation in exposure of SEB from LPS. Since genetic expression varies with the length of exposure, a set of SEB and LPS induced time points spanning two to twenty‐four hours were investigated. Initial comparisons of over 2000 genes induced by SEB and LPS in human peripheral mononuclear blood cells were carried‐out through Microarray analysis. Of these, thirty genes were further analyzed by multiple gene quantitation techniques such as RT‐PCR and real‐Time PCR. The following genes: tripartite motif‐containing 26 (TRIM26), Homo sapiens PRP3 pre‐mRNA processing factor 3 homolog (yeast) (HPRP3P), coatomer protein complex subunit gamma 2 (COPG2), and Homo sapiens ST6 (alpha‐N‐acetyl‐neuraminyl‐2,3‐beta‐galactosyl‐1,3)‐N‐acetylgalactosaminide alpha‐2,6‐sialyltransferase 6 (ST6GALNAC6), contain functions such as DNA binding, transportation from ER to Golgi network, ATP binding, inflammation and apoptosis as well as unique to the gene expression profile of each toxin. The multiple conformational analyses utilized in this study will not only allow us to confirm the results obtained through Microarray experiments but also to better understand the various mechanisms and pathways induced by both SEB and LPS.
Polymerase delta and epsilon proofreading cooperate with MMR to suppress lethal mutations and cancer in mice
Authors: Albertson Tina M. , Hays Laura E. , Bugni James M. , Ogawa Masanori , Treuting Piper M. , Goldsby Robert E. , Preston Bradley D.
Published: June 2021

Studies in yeast show that mismatch repair (MMR) and DNA polymerase (Pol) proofreading cooperate to ensure faithful DNA synthesis. However, the functional relationship of these pathways in mammals is not known. We recently reported that mice harboring defects in Pol δ proofreading (Pold1e), Pol ε proofreading (Polee) or MMR (Mlh1Δ) have distinct mutator and cancer phenotypes (PNAS 106:17100, 2009). Intercrosses of Pol δ ‐, Pol ε‐, and MMR‐mutant mice showed that Pol δ and ε proofreading act in parallel pathways to prevent spontaneous mutation and cancer. In contrast, proofreading and MMR exhibited a synergistic relationship. The combined loss of proofreading and MMR was embryonic lethal; Pold1e/eMlh1Δ/Δ embryos died early (E6–9) whereas Polee/eMlh1Δ/Δ died late (E15–20) in development. Mice heterozygous for proofreading and nullizygous for MMR (Pold1+/eMlh1Δ/Δ or Pole+/eMlh1Δ/Δ) were viable, but died of lymphoma and medulloblastoma before 3 months of age. Pole+/eMlh1Δ/Δ animals had accelerated cancer mortality compared to Pold1+/eMlh1Δ/Δ mice and a higher neonatal mortality. Pold1+/eMlh1Δ/Δ cells exhibited a “super‐mutator” phenotype, with spontaneous mutation rates 1000‐fold over wild‐type cells. These studies show that Pol δ and Pol ε proofreading act independently of one another and each cooperates with MMR to prevent lethal mutations and cancer in mice.
MAP kinase‐mediated regulation of DNA replication origin licensing and Cdt1 stability
Authors: Cook Jeanette Gowen , Chandrasekaran Srikripa , Tan Ting Xu
Published: June 2021

Eukaryotic cells employ many mechanisms to ensure that their genomes are completely duplicated precisely once each cell cycle. One of the most important of these mechanisms is the regulation of origin licensing by the chromatin loading of the MCM complex through the concerted action of ORC, Cdc6 and Cdt1. Origin licensing control must be maintained even when cells are challenged with extracellular perturbations such as DNA damage or agents that trigger a cellular stress response.

We have investigated effects on the origin licensing system mediated by the cellular stress response which is induced by a variety of insults such as hyperosmotic shock, exposure to bacterial toxin, or inflammatory cytokines. Unlike the effects of DNA damage, both Cdt1 and Cdc6 are stable during a cellular stress response; in fact Cdt1 is hyperstable and is no longer a substrate for DNA damage‐induced degradation. Instead, Cdt1 is robustly phosphorylated by the stress‐activated p38 and JNK members of the MAP kinase family. We further find that the activity of both p38 and JNK MAP kinases not only prevent de novo MCM chromatin loading during G1, but also induce rapid unloading of MCM complexes. The outcome of ongoing experiments with stably‐expressed mutationally altered forms of Cdt1 will shed light on these novel molecular consequences of stress kinase signaling.

Supported by NIH GM083024 and CA094907.
Expression profiling of cellular transformation induced by tumor‐associated DNA polymerase beta variant I260M
Authors: Donigan Katherine Ann , Sweasy Joann
Published: June 2021

DNA damage in vivo is estimated to occur upwards of 20,000 lesions per cell daily. Base excision repair (BER) resolves abasic sites and damaged bases. During BER, DNA polymerase β (Pol β) fills DNA gaps after excision of damaged bases. Approximately 30% of human tumors express Pol β variants not present in normal tissue. Many have altered fidelity and activity in vitro and induce cellular transformation. Prostate tumor variant I260M transforms cells and is a sequence‐context dependent mutator. To test the hypothesis that mutations induced in vivo by I260M lead to transformation, we characterized the genome‐wide expression profile of a clone expressing I260M compared to its non‐induced counterpart. Using a 1.5 fold cutoff with a false discovery rate of <0.05, 912 genes were found to be altered. Gene Ontology clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. Three nodes of interest with unaltered expression exhibited dysregulation of downstream targets. One node, peroxisome proliferator activator protein γ (PPARG), was found to have a coding mutation present only in transformed cells. PPARG is a transcription factor implicated as a tumor suppressor. This mutation may have contributed to driving cellular transformation. We conclude that I260M induces cellular transformation by a mutational mechanism.

This work was supported by 5R01CA080830 to J.S.
MYC is a Target for microRNA‐33b
Authors: Agha Apana Agha Louis , Li Yong
Published: June 2021

c‐Myc is a basic helix‐loop‐helix zipper protein which heterodimerizes with Max, binds DNA, and modulates expression of a specific set of target genes. The deregulation of this gene is one of the most common abnormalities found in human cancers and other diseases like cardiovascular anomalies. In recent years, microRNAs (miRNAs) have emerged as tiny master regulator of most genes and research in the last two decades focuses on identifying miRNA target genes. miRNAs are endogenous 20–25nt short RNAs that play important gene‐regulatory roles by binding to the 3′‐UTR of their target gene to direct their posttranslational repression. In this study we aim to determine whether miRNAs directly regulate MYC expression and its functions. Using a Luciferase‐dependent reporter screening assay, we have determined that miR‐33b profoundly regulates c‐myc through direct binding to the 3′‐UTR of the MYC gene. Real‐Time PCR data suggests that when miR‐33b is over‐expressed, MYC mRNA is significantly down‐regulated. Furthermore, we revealed that over‐expression of miR‐33b down‐regulates c‐Myc and its target protein, and adversely affects cell processes such as cell proliferation, cell cycle, cell transformation and cell migration. In summary, our findings strongly support two major aspects: miR‐33b is involved in targeting MYC and hence affects c‐myc function and miRNAs could act as potential targets to develop novel therapies against various types of cancers.
Mechanism of down regulation of RNA polymerase III‐transcribed ncRNA genes in macrophages by Leishmania
Authors: Rana Tanu , Misra Smita , Mittal Mukul K , Farrow Anitra L , Chaudhuri Gautam
Published: June 2021

Most of the non‐coding RNA (ncRNA) genes including those of several miRNAs are transcribed by RNA polymerase III (Pol III). The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non‐coding RNA genes in the macrophages to favor the establishment of their infection in the phagolysosomes of these cells. We report here the potential mechanism of these down regulations. We found that the ncRNA genes that are repressed by Leishmania infection in macrophages contain a ‘B‐box’ at their promoters and thus require the Pol III transcription factor TFIIIC110 for their expression. Leishmania promastigotes through their surface protease activate the thrombin receptor PAR1 in the macrophages. This activation of PAR1 increases the levels of cytosolic Ca2+ which in turn activates the Ca2+‐dependent protease calpain 1. Calpain 1 then degrades TFIIIC110 to inhibit the expression of the selected ncRNA genes. Immunofluorescence microscopy showed that PAR1 is abundantly expressed on the surface of the macrophages. Avirulent stock of Leishmania does not express surface gp63 and could not down regulate ncRNAs in the exposed macrophages. Inhibition of PAR1 or calpain 1 in macrophages made them relatively resistant to Leishmania infection. Supported by NIH NIAID grants R01AI042327 and R21AI076757 to GC.
Identification and characterization of a thiamine pyrophosphate riboswitch in Mycobacterium tuberculosis: A new drug target in tuberculosis drug design
Authors: Mitra Sunayana , Aboul‐ela Fareed
Published: June 2021

Riboswitches are cis‐acting mRNA regulatory elements which control the gene activity by binding to small ligands such as nucleotides, amino acids etc. This also makes them an attractive drug target. The thiamine pyrophosphate (TPP) binding riboswitch is involved in the control of the thiamine biosynthetic pathway in E.coli which is essential for its survival. Similar regions were identified in Mycobacterium tuberculosis, the causative organism of the disease tuberculosis, through sequence alignment studies. However, these putative riboswitches remain to be structurally and biochemically characterized. Analysis of one such candidate, the ThiC riboswitch by in‐vitro structure probing processes such as NMR and selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) combined with computational biology methods reveal a probable riboswitch which is tested for its binding ability to thiamine and drug‐like thiamine analogs in‐vitro by above schemes. It is also tested in‐vivo by GFP tagging and cloning into E.coli, where fluorescence is used as an indicator for riboswitch activity. This two pronged approach gives insights into its mechanism of action, opening new horizons in drug design and development. This research is funded by NIH/NCRR(P20RR020159)
Identifying IRES RNA‐Protein Complexes by SILAC based Mass Spectrometry
Authors: Tsai Becky Pin , Wang Xiaorong , Huang Lan , Waterman Marian L
Published: June 2021

The dynamic nature of RNA protein complexes has made it challenging to study post‐transcriptional regulation in development and disease. We have developed a strategy to rapidly isolate and characterize in vivo assembled protein complexes for specific RNAs. Our proof‐of‐principle RNA target is a 1.2 kb IRES (internal ribosome entry site) in the 5′UTR region of Lymphoid Enhancer Factor‐1 (LEF1) mRNA. IRESes provide an alternative mechanism of ribosome recruitment and translation that is not well understood. To isolate in vivo assembled IRES‐protein complexes, the LEF1 IRES is engineered with a cluster of stem‐loops recognized by in vivo biotinylated MS2 bacteriophage coat proteins. The MS2 protein is used to rapidly isolate the complex with magnetic streptavidin beads.

Our strategy is designed to isolate in vivo assembled RNA‐protein complexes for LC MS/MS identification of IRES binding proteins. SILAC‐based quantitative MS is used to detect proteins enriched on the target LEF1 IRES vs. non‐IRES mRNA. Results include known and putative IRES binding factors that were moderately enriched on the LEF1 IRES. The finding suggests that IRES elements assemble complexes of canonical RNA regulatory factors, rather than a few dedicated factors. In summary, our method provides a versatile and highly adaptable approach that can be applied to different RNA‐protein interactions.
Structural insights into mechanisms of the small RNA methyltransferase HEN1
Authors: Huang Ying , Ji Lijuan , Huang Qichen , Vassylyev Dmitry , Chen Xuemei , Ma Jin‐Biao
Published: June 2021

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of approx 20–30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi‐interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2′‐O‐methylation on the 3′ terminal nucleotide. A conserved S‐adenosyl‐l‐methionine‐dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 Å crystal structure of full‐length HEN1 from Arabidopsis in complex with a 22‐nucleotide small RNA duplex and cofactor product S‐adenosyl‐l‐homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2′ and 3′ hydroxyls on the 3′‐terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+‐dependent 2′‐O‐methylation mechanism.
Biophysical characterization of features of RNA helicase A that confer translational control of retroviral and selected cellular mRNAs
Authors: Ranji Arnaz K , Kvaratskhelia Mamuka , Boris‐Lawrie Kathleen A
Published: June 2021

The 5′ untranslated region of some retroviruses and cellular mRNAs contain a cis‐acting Post‐transcriptional Control Element (PCE) that is necessary for efficient translation. Specific interaction of PCE with host RNA helicase A (RHA) is necessary for translation of the PCE‐containing viral and cellular mRNAs. This study sought to understand features of RHA necessary for selective interaction with the RNA switch. Site‐directed mutagenesis determined that the central DEIH helicase core is necessary for PCE activity, while the RGG domain is necessary for the nuclear localization. Here biophysical analyses tested the hypothesis that two redundant N‐terminal RNA binding domains are necessary to confer specific interaction with the Spleen Necrosis Virus (SNV) and junD PCE target RNA. Protein footprinting was employed to detect amino acids of RHA that interact with PCE RNA. Electrophoretic mobility shift assays and fluorescence polarization assays determined that the N‐terminal double‐stranded RNA binding domains specifically recognize structural features of PCE RNA. Loss‐of‐function point mutations in the double‐stranded RNA binding domains abolished binding to target mRNAs. The DEIH core does not exhibit affinity for the target mRNA, while the C‐terminal RGG domain binds non‐specifically to PCE RNA. Our results posit the model that PCE interaction with the N‐terminal domain of RHA exposes the DEIH domain, which catalyzes RNP rearrangement that facilitates polysome association. Molecular dissection of this new translational control axis may provide strategic drug targets for infectious disease and cancer.

Funding: Glenn Barber fellowship of the OSU College of Veterinary Medicine and NIH R01 CA108882 and P01CA00730
Fatty Acid Suppression of Cell Proliferation is Associated with the Inhibition of Polyisoprenylated Methylated Protein Methyl Esterase
Authors: Amissah Felix , Taylor Shalina , Duverna Randolph , Lamango Nazarius S
Published: June 2021

Protein polyisoprenylation is essential for the activities of oncogenic proteins such as Ras. The final, reversible step of polyisoprenylation pathway is catalyzed by an ester forming polyisoprenylated protein methyl transferase and polyisoprenylated methylated protein methyl esterase (PMPMEase). Various free fatty acids (FFAs) were analyzed to test whether their beneficial health effects are associated with PMPMEase inhibition. The potent inhibitory FFAs were further analyzed for their effects on cell viability. Arachidonic acid (AA) and lauric acid (LA) inhibited PMPMEase with 0.19 and 6.5 ìM, respectively. AA was over 63‐fold more inhibitory than Prostaglandin A2 (Ki of 12 ìM) while prostaglandin E2 and long chain saturated fatty acids were not inhibitory even at 1 mM. AA and LA inhibited the viability of human neuroblastoma cells with EC50 values of 11 and 25 ìM respectively, after 72 h exposure. Therefore AA and other PUFAs may be involved in the regulation of cell proliferation and that the elevated levels of COX‐2 in tumors convert PUFAs which inhibit PMPMEase at physiological concentrations, to prostanoids that are ineffective at inhibiting both PMPMEase and thus cell proliferation. NSAIDs may thus owe their anticancer effects through their ability to protect the PMPMEase‐ and cell proliferation‐inhibiting PUFAs from COX‐2 metabolism.

Grant Funding Source: NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020)
Cyclin D1 regulates hepatic lipid metabolism
Authors: Hanse Eric Allan , Mullany Lisa K , Mashek Mara , Mashek Douglas G , Albrecht Jeffrey H
Published: June 2021

Cyclin D1 regulates progression through the G1‐S phase transition of the cell cycle and plays an important role in liver regeneration. Cyclin D1 may also effect other cellular functions including metabolism. Here, we examined the role of cyclin D1 in hepatic liver fatty acid metabolism.

METHODS

Mice were transfected with adenoviral vectors encoding cyclin D1 and cyclin D1‐KE, a mutant which does not induce cell cycle progression. In addition, primary rat hepatocytes and the differentiated mouse hepatocyte cell line AML12 were transfected with the same adenoviral vectors or siRNA targeting cyclin D1.

RESULTS

Cyclin D1 regulated the expression of many mRNAs associated with lipid metabolism including downregulation of genes involved in lipogenesis (Srebplc, Fasn, Spot14 and Pklr). In cultured primary hepatocytes, overexpression of cyclin D1 (but not D1‐KE) led to decreased de novo lipogenesis and fatty acid incorporation into triglycerides and phospholipids. Cyclin D1 also promoted fatty acid oxidation and increased the import of fatty acids into hepatocytes. Cyclin D1 also inhibited the activity of Srebplc, Ppara, and Chrebp reporter constructs, whereas siRNA to cyclin D1 increased their activity in vitro.

CONCLUSION

These studies suggest that cyclin D1 regulates lipid metabolism in the liver, potentially through downregulating Srebp1c, Ppara and Chrebp activities.