bluuebunny/crossref_metadata_2025_split huggingface dataset:
CpG Methylation Inhibits Prolidase Promoter Driven Transcription
Authors: Lanaghan Zeljka Miletic , Eni‐aganga Ireti , Balasubramaniam Muthukumar , Dash Chandravanu , Pandhare Jui 
Published: June 2021

Prolidase, a cytosolic exopeptidase, and a member of the matrix metalloproteinase (MMP) family, is encoded by the PEPD gene in humans. Since Prolidase is the only enzyme which can cleave dipeptides containing C‐terminal proline, or hydroxyproline, it plays an essential role in protein metabolism, collagen turnover, and matrix remodeling. Prolidase is essential in several physiological and pathological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Surprisingly, little is known about the molecular and cellular regulation of PEPD gene expression. In initial studies, we observed a dense CG‐rich portion around the transcription start site, suggesting a possible role of promoter methylation in the transcriptional regulation of PEPD gene. To study the transcriptional regulation of PEPD gene, we cloned and characterized the human PEPD promoter. The PEPD promoter was amplified from the human genome via PCR and inserted into a luciferase reporter construct. As demonstrated in our results, increase in luciferase activity was obtained after transfection of the PEPD luciferase reporter in HEK293T, indicating the successful construction of the luciferase reporter. Next, we performed in vitro methylation of the PEPD promoter using SssI methyltransferase which methylates DNA CpG islands. Methylation was confirmed by BstUI digestion that cleaves at unmethylated CpG dinucleotides but not at methylated CpG. The CpG methylation significantly inhibited PEPD promoter activity in comparison to the unmethylated construct. Taken together, we believe, these results will generate new knowledge on the molecular regulation of prolidase and provide better insight in our understanding of regulation of prolidase expression in various physiological and pathological conditions.Support or Funding InformationSupported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the gene regulation of Proteus vulgaris Rts1 HigB‐HigA toxin‐antitoxin system
Authors: Zaldana Kimberly Silvia , Pavelich Ian , Moller Abraham , Dunham Christine M. 
Published: June 2021

Bacterial toxin‐antitoxin systems are abundant genes that modulate a beneficial phenotypic switch to shift cells' focus towards survival in times of stress, including antibiotic exposure. During normal growth, toxin‐antitoxin complexes transcriptionally repress their own expression by binding at DNA operator regions that overlap with their own promoters. Toxin binding to the antitoxin‐DNA complex is important because it can result in increased repression. Upon stress, including nutrient limitation and antibiotic exposure, antitoxins are proteolyzed, releasing the toxin component. Released toxin prevents further growth by inhibiting replication, translation or cell wall synthesis. Given these important roles, it is critical to understand how toxin‐antitoxin systems regulate this phenotypic switch between a growth and dormant state that is antibiotic tolerant. The HigB‐HigA protein‐protein complex was first identified on the multi‐drug resistant Rts1 plasmid from Proteus vulgaris(Pv) associated with a post‐operative urinary tract infection. This toxin‐antitoxin complex is not responsive to antibiotics suggesting this aids in tolerance to antibiotics. HigA binding to HigB neutralizes the toxic effects of HigB and forms a heterotetramer. Most toxin‐antitoxin systems encode the antitoxin first and the toxin gene second, resulting in higher antitoxin expression to suppress toxin. However, the higB‐higA gene is unique in that its gene order is reversed with the higB toxin encoded first. Additionally, there are two putative promoters and, although it is clear that the main promoter is active (Phig), the activity of the second internal promoter (PhigA) that precedes the higA antitoxin is unknown although the antitoxin should be expressed to high levels. I hypothesize that PhigA is a strong promoter that results in high expression of the HigA antitoxin. I also hypothesize that the Phig promoter is weak, resulting in lower toxin and antitoxin expression. Here, I determine that unexpectedly, mutation of both the −35 and −10 sites, the −35 site or the −10 site causes an increase in beta‐galactosidase activity as compared to the wild‐type. These data suggest that the weak internal promotor (PhigA) is more active in regulating HigA antitoxin expression. Future work will explore how modulating the operator sites influences expression and will determine how transcriptional control may control HigB‐HigA expression.Support or Funding InformationThis research is funded by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award 1015487 (to CMD).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of histone chaperone Nucleophosmin‐mediated transcriptional regulation in oral tumorigenesis
Authors: Dey Suchismita , Senapati Parijat , Bhattacharya Aditya , Sudarshan Deepthi , Das Sadhan , Kumar Manoj , Kodaganur Gopinath S. , Maleikal Tessy Thomas , Kundu Tapas K. 
Published: June 2021

Histone chaperones regulate the histone traffic in the cell and are involved in different cellular processes including transcription regulation. Nucleophosmin (NPM1), a histone chaperone, enhances acetylation‐dependent chromatin transcription. Acetylation of NPM1 by lysine acetyltransferase (KAT) p300 further enhances its histone chaperone activity and transcription activation potential. Moreover, the acetylated pool of NPM1 (AcNPM1) colocalizes with RNA Polymerase II foci in the nucleus, indicating its role in transcription. NPM1 is also a potent inducer of autoacetylation of p300 thereby enhancing its KAT activity. In the context of cancers, NPM1 is overexpressed in several solid tumors including oral squamous cell carcinoma and regulates genes implicated in oral cancer. AcNPM1 levels are also elevated with increasing grades of oral cancer. However, the precise role of AcNPM1 and the mechanism of transcription regulation by NPM1 are unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP‐seq) to identify the genome‐wide localization of AcNPM1 on chromatin. Interestingly we find that AcNPM1 is enriched on promoters of genes that are co‐occupied by RNA Polymerase II and other histone modifications related to active transcription. RNA‐sequencing after NPM1 knockdown in an oral cancer cell line revealed that NPM1 regulates the expression of genes involved in oral tumorigenesis. Inducible knockdown of NPM1 in an orthotopic mouse model also validates our finding that NPM1 indeed is involved in oral cancer manifestation. These results provide insights into the gene network regulated by NPM1 and AcNPM1 and the possible role of its overexpression and hyperacetylation in oral cancer. In the same context, the underlying mechanisms of transcriptional activation by NPM1/AcNPM1 and the role of NPM1 in the process of tumorigenesis are also currently under investigation.Support or Funding InformationJawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Sir JC Bose Fellowship, Department of Science and Technology (DST), India (Grant No. SR/S2/JCB‐28/2010), Department of Biotechnology (DBT), India (Programme Support on ‘Chromatin and Disease’, Grant No. BT/01/CEIB/10/III/01 and Virtual National Oral Cancer Institute, Grant No. BT/PR17576/MED/30/1690/2016)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of mitochondrial DNA transcription by protein post‐translational modifications
Authors: Dittenhafer‐Reed Kristin E 
Published: June 2021

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome, including additional oxidative phosphorylation subunits, and the proteins necessary for mtDNA replication, expression, and stability, is encoded by the nuclear genome. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible post‐translational modification of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. Here, using in vitro biochemical and cell biology approaches, we describe the effects of lysine acetylation and serine/threonine/tyrosine phosphorylation on the mtDNA binding ability and transcriptional activity of three proteins, mitochondrial transcription factor B2 (TFB2M), the mitochondrial polymerase (POLRMT), and mitochondrial ribosomal protein L12 (MRPL12). Our in vitro biochemical results indicate that phosphorylation of TFB2M may impede mtDNA binding and subsequently alter transcription initiation. Modification of MRPL12 may alter the ability of MRPL12 to bind and stabilize POLRMT. We also report a temporal response in the transcript level of mitochondrial and nuclear genes in response to cell growth conditions used to drive a dependence on mitochondrial metabolism. Importantly, mtDNA content is unchanged in these conditions, providing evidence for a regulated transcriptional response of the mitochondrial genome. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Analysis of RNA Exosome Mutants Linked to Disease Using a Saccharomyces cerevisiae Model System
Authors: Enyenihi Liz Eno , Corbett Anita H , Joshi Samika , Leung Sara , Hess Laurie , Sterrett Maria , Basrai Munira , van Hoof Ambro , Fasken Milo B 
Published: June 2021

The RNA exosome complex is a key component of RNA processing and quality control that both degrades and processes many classes of RNA. This complex is highly conserved among eukaryotes and was first identified and studied in budding yeast (S. cerevisiae). Mutations in the human EXOSC2 gene, which encodes a cap subunit of the RNA exosome, have been linked to a novel syndrome characterized by retinitis pigmentosa, progressive hearing loss, premature aging, short stature, mild intellectual disability and distinctive gestalt. While the amino acid substitutions in EXOSC2 that cause this syndrome are known, how these amino acid changes impact RNA exosome function is not. The goal of my project is to analyze the functional consequences of retinitis pigmentosa‐linked amino acid substitutions modeled in the budding yeast ortholog of EXOSC2, Rrp4.The two variants I have analyzed, rrp4‐G58V and rrp4‐G226D, correspond to patient mutations G30V and G198D, respectively. I first assessed growth of the mutant strains compared to wildtype yeast cells, which revealed that rrp4‐G226D mutant cells exhibit a growth defect at 37°C, whereas the rrp4‐G58V mutant cells grow normally. To assess whether these amino acid substitutions affect Rrp4 protein levels, I used immunoblotting. Results of this analysis reveal that the rrp4‐G58V and rrp4‐G226D proteins are expressed, but at somewhat reduced level compared to wildtype Rrp4. In the future, I will continue characterization of the mutants by using biochemical approaches to study the assembly of the RNA exosome complex and genetic analysis of rrp4 mutant interactions with RNA exosome cofactors.Support or Funding InformationEMORY INITIATIVE TO MAXIMIZE STUDENT DEVELOPMENTEmory Initiative to Maximize Student Development: R25 GM125598Neurodevelopmental Role of an RNA Binding Protein Required for Cognitive Function: R01 MH107305This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Mutations in the DEXD‐motif of the Yeast RNA‐dependent ATPase Sub2
Authors: McCann Jack Thomas , Hansen Sarah , Hoskins Aaron 
Published: June 2021

Pre‐mRNA splicing by the spliceosome is an essential part of eukaryotic gene expression. Errors in splicing can be detrimental to an organism, which is why it is extremely important to understand the spliceosome's mechanisms of assembly and action. Key features of splicing include the numerous RNA‐protein remodeling and RNA unwinding reactions that aid in spliceosome assembly. Some of these reactions require one of three, well‐conserved DEAD‐motif proteins that are part of the splicing machinery (Prp5/DDX46, Prp28/DDX23, and Sub2/UAP56). While Prp5 and Prp28 contain the canonical DEAD tetrapeptide motif within their active site, Sub2 is unusual in that it instead contains a conserved DECD motif. The goal of my research is to understand the function of the cysteine within Sub2's DECD motif in vitro and in vivo. Contrary to previous reports, I have shown that yeast containing a DECD to DEAD mutation in Sub2 are viable with little impact on yeast proliferation. To explain the conservation of Sub2's DECD sequence, we looked for changes in yeast phenotypes under stress conditions. I exposed yeast containing either DEAD‐Sub2 or DECD‐Sub2 to hydrogen peroxide and found that yeast containing DECD‐Sub2 died at lower oxidative stress conditions than the DEAD mutant. This suggests that Sub2's DECD‐motif cysteine is capable of being oxidized and that this results in a disruption in gene expression. Since one cysteine oxidation product (sulfinic acid) is isosteric with aspartate, I am now testing a DEDD mutant of Sub2 for function as well as attempting to chemically characterize the amino acid modifications resulting from Sub2 oxidation. Together my results will provide insight into a potential function for Sub2's conserved DECD‐motif cysteine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioconjugates of Protein A to Gold Nanoparticles
Authors: Lee Christina , Naing Tiffany , Herrera Daisy , Herrera Natalie , Hoang Audrey , Marin Melinda 
Published: June 2021

Gold nanoparticles (GNPs) possess rich surface properties with low toxicity that allow it to interact with and participate in conjugation of various biomolecules. As proof of principle, gold nanoparticles were synthesized to a spherical shape with a diameter between 20–30 nm and conjugated to protein A of Staphylococcus aureus bacteria. Conjugation protocols were optimized whereas any size or shaped nanoparticle can be conjugated to any biomolecule. Applications of this technique can be utilized in biomedical imaging techniques to offer more optical contrast and visibility of tagged molecules such as in Magnetic Resonance Imaging (MRI), Chronic Traumatic Encephalopathy (CTE) or to create gold Nano shells in the detection and treatment of cancer cells. GNPs can be synthesized into Nano shells, conjugated to an antibody using the conjugation protocols reported and infused into cancer cells. The gold nanoshells are able to absorb near infrared light, which excites the electrons and, in an attempt, to return to a lower energy, release heat to the external environment. As a result, cancer cells that are also attached receives this heat and are destroyed. The optimization of the nanoparticles to biomolecules will allow for nanomedicines described to become reality.Support or Funding InformationNIH BUILD PODER Grant Award Number RL5GM118975This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Regulation of Cellular Proliferation by VACM‐1/CUL5 is Dependent on its Posttranslational Modifications by NEDD8
Authors: Sundquist Skylar Paige , Lee Si Eun , Burnatowska‐Hledin Maria 
Published: June 2021

VACM‐1/CUL5 acts as the scaffold protein in the E3 ligase complex in the ubiquitin‐dependent protein degradation pathway. The overexpression of VACM‐1/CUL5 is known to inhibit proliferation, whereas inhibition of VACM‐1/CUL5 expression induces cellular proliferation. Thus, VACM‐1/CUL5 is implicated in cancer pathways. The effect of VACM‐1/CUL5 on cellular proliferation is dependent on its post‐translational modification (PTM) by NEDD8 protein (neddylation). The relationship between NEDD8 and VACM‐1/CUL5 is important for cell cycle regulation and offers a target for cancer therapy. This work explores the structure‐function relationship between VACM‐1/CUL5 and its neddylation. VACM‐1/CUL5 was mutated at the putative neddylation site Lysine (K) 724 and at three potential neddylation sites, K724, K727, and K728 (3K mutant). Our work suggests that the expression of the K724 mutant induces growth whereas expression of the 3K mutant does not affect cell growth. Interestingly, Western Blot analysis indicates that the 3K VACM‐1/CUL5 mutant is still neddylated, which suggests that VACM‐1/CUL5 may be neddylated at additional lysine sites. Our current work focuses on characterizing cells transfected with the mutated VACM‐1/CUL5 cDNAs to elucidate how site‐specific neddylation may control alternative signaling pathways.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neurodegeneration‐related amyloids interact with the prion protein
Authors: De Cecco Elena , Narkiewicz Joanna , Grandolfo Micaela , Parisse Pietro , Legname Giuseppe 
Published: June 2021

Tau protein is the major component of intracellular aggregates that characterize a number of neurodegenerative disorders collectively called tauopathies. Tau amyloids propagate from cell to cell through different routes, such as dynamin‐dependent endocytosis, actin‐dependent macropinocytosis, tunneling nanotubes. Here, we evaluate the role of the prion protein PrPC in promoting the internalization of synthetic tau aggregates in cultured cells. As previously found by our group, the prion protein facilitates the uptake of synuclein fibrils in neuroblastoma cell lines, in primary neurons and in mice. Using confocal microscopy, we show that also tau fibrils take advantage of the presence of the prion protein on the plasma membrane to gain entrance to the cell, and that antibodies directed against the binding site of the fibrils on PrPC can decrease the internalization of the aggregates. Moreover, tau fibrils interact with both the cellular form PrPC and the pathological conformer PrPSc, inducing clearance of the latter through a combination of lysosomal activation and inhibition of prion conversion. Our results indicate that the prion protein can act as a receptor for aggregated proteins (synuclein, tau), and that targeting PrPC could be a therapeutic strategy to slow down the spreading of tau pathology.Support or Funding InformationThis work was supported by the Program FIRB (Prot. RBAP11FRE9_001).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic analysis of the poly‐ubiquitin profiles in Alzheimer's Disease
Authors: Zhang Tian T , Dammer Eric B , Abreha Measho , Duong Duc M , Lah James J , Levey Allan I , Seyfried Nicholas T 
Published: June 2021

Several neurodegenerative diseases including Alzheimer's Disease (AD), have characteristic ubiquitin positive pathological protein aggregates. Currently, the specific ubiquitylation protein substrates associated with these aggregates in AD brain is poorly understood. We analyzed control and AD postmortem brain tissues to examine the changes in the proteome and ubiquitylation substrates associated with AD pathology. Affinity capture using tandem ubiquitin binding entities (TUBEs) was utilized to enrich poly‐ubiquitylation substrates from AD and age‐matched control postmortem brain tissues (n=3 each group). Label‐free mass spectrometry (MS) based proteomic analysis identified a total of 2112 unique proteins across all samples and bead controls. After controlling for non‐specific binding, we identified 197 proteins that were at least two‐fold enriched in AD compared to control cases. Some of these targets include ubiquitin, Tau, p62 sequestosome and 14‐3‐3 proteins, which are known to associate with AD neuropathology. A cross comparison with previous proteomic studies revealed that 108 of those 197 proteins have increased site‐specific levels of ubiquitylation in AD brain. Tau poly‐ubiquitylation from AD brain homogenates was confirmed by affinity capture using either pan or linkage‐specific (Lys48 and Lys63) poly‐ubiquitin TUBEs and immunoblotting. These findings show the utility of MS to map ubiquitylated substrates in the human brain and provide a better understanding of the mechanisms underlying post‐translational modifications of pathological proteins in AD.Support or Funding InformationNational Institute on Aging (U01AG046161‐02, R21AG054206, 5R01AG053960, RF1AG057470, and RF1AG057471) and the NINDS Emory Neuroscience Core (P30NS055077).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.