bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ate1‐mediated posttranslational arginylation is essential for stress response regulation and mutagenesis suppression
Authors: Kumar Akhilesh , Birnbaum Michael D , Morgan William , Zhang Fangliang 
Published: November 2023

Arginylation is a posttranslational modification which leads to addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence and the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in these conditions. In our study by using multiple types of eukaryotic cells as test models, we found that Ate1/arginylation is essential for stress response, apoptosis and mutagenesis suppression. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of stressing factors including heat shock, oxidative and osmotic stress, heavy metal and radiation. We demonstrated that Ate1 and its arginylation activity are up‐regulated during stress and activate the intrinsic apoptotic pathway with dependence on the mitochondria‐related apoptosis‐inducing factor (Aif1), but not directly on the ubiquitin‐proteasome system or Caspase (Yca1). Moreover, over‐expression of Ate1 also leads to cell death which depends on its enzymatic activity. Furthermore, we found that Ate1/arginylation is required to suppress mutagenesis in the population during DNA damaging stress. Our data thereby explain the basis for the involvement of arginylation in physiological and diseased conditions by its regulation of cell death and/or mutagenesis. We also uncover a novel mechanism by which a posttranslational modification exerts a global effect on mutagenesis.Support or Funding InformationNIH R01GM107333DoD/CDMRP PC140622 and PC141013ACS IRG‐98‐277‐13Sylvester Comprehensive Cancer Center Developmental Grant
Enzyme Catalyzed Expressed Protein Ligation
Authors: Henager Samuel , Chu Nam , Chen Zan , Bolduc David , Wells James , Cole Philip 
Published: November 2023

Here we show the utility of subtiligase, a previously described engineered form of the bacterial protease subtilisin that has been converted to a peptide ligase, for creating semisynthetic proteins from intein‐generated protein thioesters and synthetic peptides. Expressed protein ligation (EPL) is an established method that exploits a chemoselective (‘native chemical’) ligation between a recombinant protein thioester generated via an intein and an N‐Cys containing synthetic peptide. While a powerful method for protein semisynthesis, EPL is limited by its standard requirement for a Cys residue at the ligation junction and sometimes sluggish reaction rates. In this study, we investigate both ubiquitin and glutathione S‐transferase as model protein thioesters to assess the efficiency of subtiligase‐mediated protein ligation with respect to sequence selectivity proximal to the ligation junction. Many amino acid sequences at the junction were well tolerated and led to ligation conversions as high as 50–70% in 10 minutes or less. We also showed that subtiligase could be used to produce semisynthetic tetraphosphorylated PTEN, a lipid phosphatase and key tumor suppressor gene, in its fully native form. By using this semisynthetic tetraphosphorylated PTEN as a protein standard, we could more accurately quantify the level of tail phosphorylation of cell‐derived PTEN, allowing for insights into its regulation. The application of subtiligase described here therefore offers a rapid and efficient alternative to standard EPL for protein semisynthesis.Support or Funding InformationNIH
MIF SUMOylation in regulation of its stability and activity during breast cancer progression and metastasis
Authors: Sen Progga , Zhang Xiang‐Dong 
Published: November 2023

SUMOylation is an essential posttranslational modification that regulates many cellular processes ranging from cell cycle control to protein stability. Dysregulation of SUMOylation has been implicated in various diseases including cancers. Our recent studies revealed that SUMO‐2/3 modification of macrophage migration inhibitory factor (MIF) is greatly increased in metastatic breast cancer cells compared to non‐metastatic control cells. As a pro‐inflammatory and carcinogenic cytokine, MIF acts as a direct link between inflammation and cancer. We further showed that SUMO‐2/3 modification of MIF is positively correlated with its increased levels in metastatic cells compared to non‐metastatic cells, whereas inhibition of SUMOylation by overexpression of SUMO‐specific isopeptidase SENP1 or SENP2 greatly decreases levels of MIF. Furthermore, we demonstrated that MIF is SUMOylated at a single lysine residue, K78, using in vitro SUMOylation assays. MIF is known to be degraded through ubiquitin‐dependent proteolysis and is ubiquitinated at K78, suggesting that MIF SUMOylation can inhibit its ubiquitination and degradation. Lastly, our protein interaction assays have revealed that MIF specifically interacts with SUMO E3 ligase PIAS3. As both PIAS3 and MIF are often upregulated in various types of cancers including breast cancer, it would be very interesting to test whether PIAS3 is the E3 ligase for MIF SUMOylation and increases MIF stability. Hence, characterization of MIF SUMOylation is expected to enrich our understanding of the cross‐talk of SUMO and ubiquitin pathways during breast cancer progression and metastasis.Support or Funding InformationWayne State University Startup GrantAmerican Cancer Society Institutional Grant
Decreasing O‐GlcNAcylation of 8‐oxoguanosine DNA Glycosylase (OGG1) in Cardiac Myocytes Cultured in High Glucose Leads to Improvement of DNA Repair and Mitochondrial Function
Authors: Cividini Federico , Dai Anzhi , Scott Brian , Diemer Tanja , Suarez Jorge , Dillmann Wolfgang 
Published: November 2023

DNA damage in cardiac myocytes resulting from increased oxidative stress is an important factor in the pathogenesis of diabetic cardiomyopathy. Although DNA repair machinery has been described in the cardiac myocyte, the regulation of oxidative DNA damage repair has been incompletely investigated. Moreover, in hyperglycemia the O‐GlcNAc post‐translational protein modification has been reported to interfere with the regular intracellular activity of metabolically relevant proteins in either an adaptive or a maladaptive manner. The enzyme 8‐oxoguanosine DNA glycosylase (OGG1) is key in DNA repair. Thus, the objective of this work was to investigate the influence of O‐GlcNAcylation of OGG1 in excessive glucose concentrations, simulating the levels observed in diabetic animals or humans. Neonatal cardiac myocytes (NCM) were isolated and cultured for 72 h in either normal glucose (NG; 5.5 mM glucose, 19.5 mM mannitol) or high glucose (HG; 25 mM glucose). The relationship between OGG1 O‐GlcNAcylation levels, enzymatic functionality and subcellular localization was analyzed. OGG1 was found to be up‐regulated and highly O‐GlcNAcylated in HG cultured NCM and was associated with increased 8‐OHdG (8‐hydroxy‐deoxyguanosine) levels, a common oxidative stress biomarker targeted by OGG1. In addition, decreased DNA quality and mitochondrial function were observed. Interestingly, OGG1 enzymatic activity in cell lysates was lower in HG compared to NG. Furthermore, in vitro experiments with recombinant OGG1 and O‐GlcNac transferase (OGT) demonstrated that O‐GlcNAcylation of OGG1 diminishes enzyme activity. We performed rescue experiments in NCM in HG by either rectifying OGG1 levels or reducing OGG1 O‐GlcNAcylation. NCM were either transduced with adenoviral particles (Adv) carrying OGG1 (Adv‐OGG1) or co‐transduced with Adv‐OGG1 and a dominant negative OGT (Adv‐dnOGT). Co‐transduced NCM showed improved OGG1 enzymatic activity compared to controls cells in spite of HG. These results were associated with lower 8‐OHdG levels. Finally, a different sub‐cellular enzyme distribution and an improvement in mitochondrial function were also detected. In conclusion, these results suggest that O‐GlcNAcylation of OGG1 could be considered a maladaptive phenomenon caused by hyperglycemia, which directly interferes with DNA repair by altering OGG1 activity and translocation, and indirectly leads to mitochondrial dysfunction.Support or Funding InformationThis work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation.
Specificity of Protein Arginine Methyltransferases (PRMTs): How Does the Trypanosoma brucei PRMT7 Limit its Activity to Monomethylation of its Target Proteins?
Authors: Jain Kanishk , Warmack Rebeccah A , Feng You , Debler Erik W , Stavropoulos Peter , Blobel Gunter , Clarke Steven G 
Published: November 2023

In the family of the nine mammalian enzymes that modify proteins by adding one or two methyl groups to the terminal guanidino nitrogen atoms of acceptor proteins, PRMT7 is unique in producing solely monomethylarginine (MMA) products. The other eight PRMTs catalyze the formation of symmetrically or asymmetrically dimethylated arginine (SDMA or ADMA) residues. Since these modifications are often recognized by reader proteins specific for MMA, SDMA, or ADMA, the biological functions of PRMTs are closely linked to the type of methylation. SDMA and ADMA residues on certain proteins have been known to up‐regulate or down‐regulate transcription, suggesting a possible regulatory function for MMA markers produced by PRMT7. In order to understand the nature of monomethylation and what keeps PRMT7 from dimethylating its substrates, we have focused our attention on the residues of its active site from the protozoan Trypanosoma brucei responsible for sleeping sickness in humans. By using site‐directed mutagenesis, we have mutated several residues including those in the “double E” loop known to interact with the methyl‐accepting substrate arginine residues and then assayed its level and type of methylation via high resolution cation exchange chromatography. Here we present results suggesting that most of these enzyme modifications result in significantly reduced activity but no change in MMA specificity. However, we have found that a mutation of glutamate‐181 (a double E residue) to an aspartate residue not only significantly diminishes enzyme activity but also confers to the enzyme the ability to asymmetrically dimethylate arginines, generating ADMA products. This result pinpoints a crucial residue in maintaining the specificity of PRMT7.Support or Funding InformationEWD was a Frey Fellow of the Damon Runyon Cancer Research Foundation. This work was also supported by grants from NIH including GM026020 (S.G.C.) and a Ruth L. Kirschstein National Service Award GM007185 (to K.J. and R.A.W.).
5hmC Distribution in Isolated Visceral Adipose Tissue Nuclei
Authors: Yu Ping , Ji Lexiang , Lee Kevin , Yu Miao , He Chuan , Ambati Suresh , Schmitz Robert , Meagher Richard 
Published: November 2023

The reprogramming of specific cell types, and in visceral adipocytes in particular, is likely to be a fundamental to obesity and its related comorbidities. We explored the hypothesis that adipose tissue contains epigenetically distinct subpopulations of adipocytes in that are differentially potentiated to record memories of their cellular environment. Adipocytes are large, fragile, and technically difficult to isolate. We developed fluorescence nuclear cytometry (FNC) and fluorescence activated nuclear sorting (FANS) of cellular nuclei from visceral adipose tissue (SsVAT) using the levels of the adipocyte‐specific protein, peroxisome proliferator‐activated receptor gamma‐2 (PPARg2). Using FANS we obtained highly purified PPARg2‐Positive (PPARg2‐Pos) nuclei, which were distinct from PPARg2‐Negative (PPARg2‐Neg) leukocyte, endothelial cell and progenitor cell nuclei. PPARg2‐Pos VAT nuclei showed 2‐ to 50‐fold higher levels of transcripts encoding most of the chromatin‐remodeling factors assayed, including those which regulate the methylation of histones and DNA cytosine (e.g., DNMT1, DNMT3A, TET2, TET3, KMT2C, SETDB1, PAXP1, ARID1A, KMT2C, JMJD6, CARM1, and PRMT5). PPARg2‐Pos nuclei have large decondensed chromatin structures. Immunofluorescence microscopy (IFM) revealed the co‐distribution of PPARg2 protein with 5‐hydroxymethylcytosine (5hmC), with nearly all nuclei staining most strongly for PPARg2 also staining strongly for 5hmC. TET‐assisted bisulfite sequencing (TAB‐seq) demonstrated 5hmCG levels were the highest for PPARg2‐High nuclei and lowest for PPARg2‐Neg nuclei across all gene regions, and 5hmC levels were remarkably dynamic in gene bodies of PPARg2‐Pos nuclei, dropping 3.8‐fold from the highest quintile of expressed genes to the lowest. In short, VAT‐derived adipocytes are epigenetically distinct with PPARg2 and 5hmC levels coordinately regulated and proportional to nuclear size.Support or Funding InformationThis work was supported by grants from UGA's Office of the Vice President for Research, UGA's Obesity Initiative and from NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, DK1003920 and DK096300) to RBM, NHGRI HG006827 grant to CH, the National Institutes of Health grant R00GM100000 to RJS, and a Patricia and Doyle Mote Biomedical Research Award to PY.
Overexpressed Gα13 stimulates Serum Response Factor mediated transcription concurrent with relocalization to a soluble fraction
Authors: McDiarmid Gray R , Tagliatela Alicia C , Meigs Thomas E. 
Published: November 2023

Heterotrimeric G proteins consist of α, β, and γ subunits, and activating, GTPase‐deficient mutations in the α subunit of several classes have been implicated in tumor progression. The G12/13 class of α subunits, comprising Gα12 and Gα13, has the unique ability to stimulate oncogenic transformation of cultured cells though overexpression of the wildtype α subunit. However, the mechanism that allows this signaling property of wildtype Gα12 and Gα13 is not understood. Using transiently transfected HEK293 cells, we examined varying levels of constitutively active and wildtype forms of Gα13 for ability to stimulate serum response factor (SRF) mediated transcription, a cell growth signaling pathway implicated in melanoma and other cancers. Constitutively active Gα13 showed an approximate 100‐fold stimulation of SRF signaling, and unexpectedly, we observed a response fully half this magnitude using wildtype Gα13. In cell fractionation experiments using an epitope‐tagged wildtype Gα13, this transiently expressed protein began appearing in the soluble fraction as its levels increased, whereas endogenous Gα13 remained in the membrane‐associated fraction. Under conditions in which Gα13 redistribution was observed, SRF activation showed a sharp increase. This stimulatory effect of wildtype Gα13 was blunted by co‐expression of Gβ1 and Gγ2 subunits, suggesting overexpressed Gα13 drives SRF signaling due to stoichiometric imbalance in the heterotrimer. Conversely, SRF stimulation by wildtype Gα12 was enhanced by these Gβ/Gγ subunits. Gβ1/Gγ2 co‐expression also changed the subcellular distribution of wildtype Gα13 to a predominantly membrane‐associated state. Furthermore, mutation of two N‐terminal Cys residues required for Gα13 palmitoylation caused localization to the soluble fraction but abolished SRF signaling, suggesting this lipid modification as critical for the soluble, overexpressed pool of Gα13 to drive this growth pathway.Support or Funding InformationWe acknowledge support from the GlaxoSmithKline and C.D. Spangler Foundations, Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
The Role of CaMKII in mGlu5 Signaling
Authors: Marks Christian Randal , Shonesy Brian C , Colbran Roger J 
Published: November 2023

Ca2+/calmodulin (CaM)‐dependent protein kinase II (CaMKII) is a serine/threonine kinase that accounts for ~1% of total protein in forebrain. Interactions of activated CaMKII with CaMKII associated proteins (CaMKAPs) and roles of CaMKII in modulating ionotropic glutamate receptors and voltage gated ion channels have been studied intensively, but the role of CaMKII in G protein coupled receptor (GPCR) regulation is less well understood. Here, we confirmed a previous report1 that CaMKII interacts with the metabotropic glutamate receptor 5 (mGlu5), a Gq coupled receptor, in vitro, in HEK293 cells and in mouse forebrain lysates. Unlike most other CaMKAPs, mGlu5a interacts with INACTIVE CaMKII, and the interaction is disrupted by Ca2+/CaM. We also found that CaMKII phosphorylates the mGlu5a C‐terminal domain (CTD) in vitro. Inclusion of the mGlu5b‐specific splice insert in the CTD does not affect the binding or phosphorylation of mGlu5 by CaMKII. We have begun to characterize CaMKII binding and phosphorylation sites in mGlu5a/b. Binding of Ca2+/CaM to mGlu5 blocks the phosphorylation of some sites on mGlu5, such that they are preferentially phosphorylated by Thr286‐autophosphorylated CaMKII, which exhibits Ca2+/CaM‐independent kinase activity. In addition, initial studies indicate that the mGlu5 CTD modulates CaMKII activation in vitro, representing a potential bidirectional regulatory mechanism. We are currently comparing CaMKII activity and subcellular localization in wild‐type and mGlu5‐null mice. Understanding the interplay between these key synaptic regulators may provide mechanistic insights into their role in the modulation of synaptic transmission.Support or Funding InformationT32‐DK007563 and AHA 15PRE25110020 to CRMR01‐MH063232 and R01‐NS078291 to RJC
Immunofluorescence Localization of Ligand‐Induced Internalization, Trafficking, and Signaling of eGFP‐Tagged Guanylyl Cyclase/Natriuretic Peptide Receptor‐A into the Subcellular Compartments
Authors: Mani Indra , Tripathi Satyabha , Garg Renu , Pandey Kailash N 
Published: November 2023

Cardiac hormone, atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which produces second messenger cGMP and lowers blood pressure and blood volume. The objective of this study was to visualize the internalization and intracellular trafficking of NPRA in the subcellular compartments of intact cells. To study the internalization and intracellular trafficking of NPRA, we utilized immunofluorescence staining and co‐immunoprecipitation (co‐IP) of plasma membrane, endosomal, lysosomal, and Rab 11 markers to follow trafficking and signaling by confocal immunofluorescence microscopy and immunoblotting. A chimeric construct of enhanced green fluorescence protein (eGFP) and NPRA (eGFP‐NPRA) was used to study internalization and trafficking of receptor in human embryonic kidney‐293 (HEK‐293) cells. The treatment of cells with ANP at different times accelerated the internalization of receptor from cell surface to cell interior. Colocalization of eGFP‐NPRA with pan‐Cadherin indicated that internalization of receptor accounted for 58%, 69%, 70%, and 75% at 5, 10, 15, and 30 min, respectively, compared with untreated control cells. Colocalization of eGFP‐NPRA with early endosome antigen‐1 (EEA‐1) marker was maximum at 5 min (63%), and gradually decreased at 10 min (43%), 15 min (31%) and at 30 min (23%) respectively. Similarly, colocalization of NPRA with lysosome‐associated membrane protein‐1 (LAMP‐1) gradually increased at 5 min (13%), 10 min (36%), 15 min (42%), and at 30 min (44%). After treatment with lysosomotropic agents, intracellular accumulation of the receptor progressively increased at 5 min and continued up to 30 min. Rab 11 was used as a recycling endosome marker, which showed that 20% receptor recycled back on plasma membrane. Co‐immunoprecipitation assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Immunofluorescence and enzyme‐linked immunosorbent assay (EIA) analyses showed a marked increase in the accumulation of intracellular cGMP concurrent with receptor internalization. Our study suggests that after ligand binding, receptor internalized and trafficked into subcellular compartments with concurrent generation of intracellular cGMP, which regulates blood pressure and cardiovascular homeostasis.Support or Funding InformationThis work was supported by NIH grants R01HL057531 and R01HL062147
Biological funtions for Ans‐linked oligosaccharides in the tethered rec‐eel luteinizing hormone
Authors: Byambaragchaa Munkhzaya , Min Kwan‐Sik , Kang Myung‐Hwa 
Published: November 2023

Japanese eel Anguilla japonica is one of the most important fish species being aquaculture in Japan but is hampered from the fact that this speices does not reproduce in captivity. Artifical induction of gonadal maturation has been successful by administration of salmon pituitary extracts, but the understanding the regulatory mechanism of gonadal development moderated by FSH and LH remains elusive due to lack of suitable amounts of eel GTHs.In the present study, we produced tethered rec‐eel LH and mutants (56, 79 and 56–79 of a‐subunit) of Asn‐linked oligosaccharides in CHO cells. In western blot analysis, its molecular weight was deteced at 32‐kDa in wild type. Elimination of the carbohydrate at a single site reduced with about 10%, where molelcular weight in double mutant decreased ove 20% compared to wild type. Now we are trying to analysis the biological activity and internalization using cells expressing eel LH/CG receptor cDNA.Support or Funding InformationThis work was supported by a scholarship form the BK21 Plus Program, the Ministry of Education, Science and Technology, Korea. This work was supported by a grant form KIMST (D11503115H480000110), Korea.
Hepatitis B virus X protein induces hepatic steatosis by enhancing the expression of liver fatty acid binding protein
Authors: Lin Xu , Wu Yun‐Li , Peng Xian‐E , Zhu Yi‐Bing , Yan Xiao‐Li , Chen Wan‐Nan 
Published: November 2023

BACKGROUND & AIMSHepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although the molecular mechanism involved in the pathogenesis of HBV‐associated hepatic steatosis still remains elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV‐producing hepatoma cells. In this study, we sought to determine the effects of Hepatitis B X protein (HBx)‐mediated FABP1 regulation on hepatic steatosis and the underlying mechanism.METHODSmRNA and protein level of FABP1 were measured by quantitative RT‐PCR and western blot. HBx‐mediated FABP1 regulation was evaluated by luciferase assay, co‐immunoprecipitation and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by Oil‐Red‐O staining and triglyceride level.RESULTSExpression of FABP1 was dramatically increased in HBV‐producing hepatoma cells, serum of HBV‐infected patients, and serum and liver tissues of HBV‐transgenic mice. Ectopic overexpression of HBx resulted in up‐regulation of FABP1 in HBx‐expressing hepatoma cells whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated FABP1 promoter in HNF3β, C/EBPα and PPARα‐dependent manner, in which HBx increased the expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other way around, knockdown of FABP1 remarkably blocked lipid accumulation both in long‐chain fatty acids treated HBx‐expressing HepG2 cells and in a high fat diet fed HBx‐transgenic mice.CONCLUSIONSFABP1 is a key driver gene in HBx‐induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα and PPARα. FABP1 may represent a novel target for treatment of HBV‐associated hepatic steatosis.Support or Funding InformationThis work was supported by the grants from National Natural Science Foundation of China (Grant number 81271822 and 81401657), State Key Project Specialized for Infectious Diseases (2012ZX10002002‐004‐006 and 2013ZX10002002‐005‐002) and Foundation of Fujian Educational Committee (Grant number JK2013021).