bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Role For RPA Phosphorylation In The Regulation Of G2 Exit In The Face Of Persistent DNA Damage
Authors: Wilson Timothy M. , Ghospurkar Padmaja L. , Haring Stuart J. 
Published: November 2023

BackgroundReplication Protein A (RPA), a heterotrimeric complex with critical roles in DNA metabolism and cell cycle progression, is observed to be hyper‐phosphorylated in response to genotoxic stress. Hyper‐phosphorylation of human RPA (hRPA) occurs primarily on the N‐terminus (NT) of the 32 kDa Rpa2 (hRpa2) subunit. In contrast, phosphorylation of yeast RPA (yRPA) on the Rpa2 (yRpa2) NT is not detectable upon exposure to commonly used genotoxic agents (e.g., methyl methanesulfonate, hydroxyurea). In fact, the yRpa2 NT has never been identified as a major site of phosphorylation upon DNA damage treatment. However, the examination of yRpa2 NT phospho‐mutants suggests that the phospho‐state of yRpa2 NT may play a key role in cell cycle progression despite the continued presence of DNA damage (checkpoint adaptation). In some instances checkpoint adaptation might allow for the positive cellular consequence of continued cell growth. This comes with a potential negative organismal cost, as checkpoint adaptation is often detrimental to cell survival and is also associated with increased genomic instability, which can ultimately lead to mutations that cause cellular disease.Study ObjectiveTo elucidate the importance of the yeast Rpa2 NT in regulating exit from the G2/M checkpoint.MethodWe examined yRpa2 phosphorylation and the cell cycle progression of yRpa2 NT mutants in an inducible HO endonuclease system that generates a single double‐strand break (DSB) that cannot be repaired by homologous recombination (HR).Results and ConclusionsWe have demonstrated that the presence of the yRpa2 NT is necessary for checkpoint adaptation. Furthermore, we have recently discovered that in the presence of a persistent lesion, yRpa2 NT hyper‐phosphorylation is now readily detected. This is consistent with our published data demonstrating that checkpoint adaptation is affected in yRpa2 phospho‐mutant cells. Because N‐terminal phosphorylation is now detectable under this condition, we have employed alanine scanning mutagenesis to identify which sites on the yRpa2 NT are phosphorylated and important for promoting checkpoint adaptation. We have also examined the kinases responsible for phosphorylating the yRpa2 NT during checkpoint adaptation. In addition, we have evidence that phosphorylation of the yRpa2 NT occurs primarily upon prolonged exposure to other agents that specifically result in DSB formation. Finally, we propose a model that suggests that Rad53 deactivation and Rfa2 phosphorylation may be linked to coordinate checkpoint adaptation in yeast. Understanding how yRPA phospho‐states influence cellular division in the presence of persistent/prolonged genotoxic stress may help provide the framework for deciphering the molecular mechanism(s) cells employ to maintain their genome.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH.
Self‐organization of zebrafish lateral line primordium morphogenesis and migration
Authors: Chitnis Ajay , Nogare Damian Dalle 
Published: November 2023

The zebrafish Posterior Lateral Line Primordium (PLLp) pioneers the establishment of the Posterior Lateral Line system. Interactions between the Wnt, FGF, Notch, and chemokine pathways in the PLLp provide a framework for understanding how cell fate and morphogenesis are coordinated in this group of about a hundred cells, as they collectively migrate under the skin from the ear to the tip of the tail. We have used a combination of experiment and computational modeling to define the mechanisms that determine both the periodic formation of neuromasts and their deposition along with inter‐neuromast cells, as the PLLp migrates along a path defined by chemokine expression. Furthermore, our high resolution imaging of the morphology and dynamics of individual cells in the primordium reveal unexpected features of how cells with distinct morphologies in distinct parts of the PLLp contribute to collective migration and communication between leading and trailing cells in the primordium. Together, our studies reveal novel features of the self‐organization of this remarkable system.Support or Funding InformationEunice Kennedy Shriver National Institute of Child Health and Human Development intramural Program
Elucidation of Saw1 and Rad1–Rad10 Recruitment to Induced Single‐stand Annealing Sites as a Function of 3′ Flap Length
Authors: Felix Juan Camberos , Fregoso Fred , Odango Rowen , Iannolo Bailey , Fischhaber Paula 
Published: November 2023

Unrepaired DNA damage leads to cancer and aging. A more detailed understanding of DNA damage repair mechanisms, specifically of double‐strand breaks (DSB's), can lead to new cancer prevention and treatment strategies. Two major DSB repair pathways include Synthesis‐Dependent Strand Annealing (SDSA), a conservative pathway resulting in perfect repair, and Single‐Strand Annealing (SSA), repair characterized by the loss of genetic material.During SSA in S. cerevisiae, complementary strands deriving from DNA repeats flanking a DSB become annealed, creating non‐homologous 3′ overhanging DNA flaps that are cleaved by the Rad1–Rad10 protein complex endonuclease activity. Prior research shows Saw1 is required to recruit Rad1–Rad10 to the DSB. However, recent studies show Rad1–Rad10 recruitment is Saw1‐independent if the overhanging flaps to be cleaved are short leaving ill‐defined the precise flap length triggering Saw1‐dependent Rad1–Rad10 recruitment.To elucidate the flap length at which Rad1–Rad10 requires Saw1 for recruitment during SSA, DSB's will be triggered by restriction digest in fluorescently labeled yeast strains containing a fluorescently‐labeled I‐SceI cut site flanked by a variable number of non‐homologous base pairs (bp) between two DNA repeats. Repair sites containing variable length overhanging flaps produced by SSA will be monitored for recruitment of Rad1–Rad10 via fluorescence microscopy in pairs of strains that are either wild‐type in SAW1 or SAW1 deletion mutants.The specialized yeast strains needed are not available and the current goal of this project is their preparation. Strains are being prepared by integration of DNA cassettes bearing the I‐SceI cut site flanked on either side by 20, 30 or 50 bp of non‐homologous DNA and the HIS3 gene. The 663 bp HIS3 gene provides the DNA repeat sequence. DNA plasmids bearing the cassettes were prepared by standard cloning methods. DNA for the 50 bp flap yeast strain has been successfully integrated into the yeast genome with PCR screens of yeast transformants showing incorporation of the correct DNA cassettes at the target chromosomal locus. Our 50 bp flap strain screen results show that the cloning scheme envisioned for the preparation of these specialized yeast strains is viable and preparation of one strain is complete.Support or Funding InformationNIH‐SC3GM093858 and the Dr. Lan K. Wong Undergraduate Research Award.
Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates
Authors: LaCava John , Molloy Kelly R. , Fenyö David , Taylor Martin S. , Chait Brian T. , Boeke Jef D. , Rout Michael P. 
Published: November 2023

LINE‐1 (L1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. L1 ribonucleoprotein particles incorporate a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against L1's mutagenic activity. We previously characterized host proteins associated with human L1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics.To build on these analyses, we have executed a suite of quantitative proteomic comparisons, yielding interactomic maps of affinity isolated L1s. These studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. We report the proteins partitioning within these distinct L1 populations and associated in vitro activities. Our observations provide a basis for the classification of L1 interactors into physical and functional modules and have enabled the development of in vitro systems to study L1 activity.Support or Funding InformationThis work was supported by NIH grants P41 GM109824, 1P50GM107632‐01, and P41 GM103314
Neuroprotective actions of hydrogen sulfide (H2S) and sulfhydration in the brain
Authors: Paul Bindu Diana , Snyder Solomon H. 
Published: November 2023

Hydrogen sulfide (H2S) has emerged as an important signaling molecule joining the ranks of the gasotransmitters, nitric oxide (NO) and carbon monoxide (CO). Analogous to NO, H2S signals via a posttranslational modification termed sulfhydration, wherein H2S mediates the conversion of –SH groups of reactive cysteine residues on target proteins to –SSH or persulfide groups. Ever since its characterization, sulfhydration has been shown to participate in a myriad of physiological processes ranging from vasorelaxation to neuroprotection. Recently we have shown that aberrant H2S metabolism and sulfhydration play key roles in the progression of neurodegeneration in Parkinson's disease (PD) and Huntington's disease (HD). In HD, mutant huntingtin disrupts the transcriptional regulatory machinery of cystathionine gamma lyase (CSE), the biosynthetic enzyme for H2S as well as its precursor cysteine. mHtt affects transcription factors responsible for expression of CSE leading to its depletion in HD. H2S also modulates the disposition of second messengers such as cGMP which results in cognitive deficits, a feature shared by several neurodegenerative disorders including PD and HD. This presentation focuses on how H2S regulates redox homeostasis via gene regulatory mechanisms, the disruption of which is a contributor to disease progression in neuronal degeneration.Support or Funding InformationRepresentative Recent Recognition/HonorASBMB/jbc Herbert Tabor Young Investigator Award (May 2015) at the third European Conference on the Biology of Hydrogen Sulfide at Athens, Greece.
BRD4 IS A HISTONE ACETYLTRANSFERASE THAT EVICTS NUCLEOSOMES FROM CHROMATIN
Authors: Ballachanda Devaiah Nanjappa , Case‐Borden Chanelle , Gegonne Anne , Hsu Chih Hao , Chen Qingrong , Meerzaman Daoud , Dey Anup , Ozato Keiko , Singer Dinah 
Published: November 2023

Bromodomain protein 4 (BRD4) is a master transcriptional and epigenetic regulator which plays a pivotal role in cancer development and immune diseases. As a chromatin binding protein that remains bound during mitosis, BRD4 has been described as a mitotic bookmark. Inhibiting its interaction with chromatin has been a successful therapeutic strategy against many cancers including acute myeloid leukemia, Burkitt's lymphoma, breast, colon and lung cancer. While BRD4 association with chromatin is known to activate transcription of key proto‐oncogenes, the mechanism involved is currently unknown. Here, we report that BRD4 has novel and intrinsic histone acetyltransferase (HAT) activity through which it acetylates histones H3 and H4 in nucleosomes. BRD4 HAT activity maps to two consensus acetyl CoA binding sites and a 40 aa catalytic site. BRD4 HAT activity is distinct from all other known HAT's with a unique lysine acetylation ‘fingerprint’ that includes acetylation of H3K4, H3K9, H3K18 and H3K27 but not H3K14. Most importantly, BRD4 acetylates H3K122, a key lysine residue critical for nucleosome stability located on the dyad axis of the nucleosome. BRD4 HAT activity and H3K122 acetylation is responsible for nucleosome eviction and opening of the chromatin, as evidenced by the ability of BRD4, but not BRD4 HAT mutants, to evict nucleosomes both in vitro and in vivo. Nucleosome clearance by BRD4 is selective and localized to the gene loci it regulates such as c‐Myc, c‐Fos and Aurora B kinase. Consistent with its role in localized chromatin de‐compaction, BRD4 HAT activity regulates transcription at these gene loci as well. BRD4 HAT activity thus plays a key role in chromatin remodeling and transcription.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Expanding the Roles of tRNA Thiolation in Bacteria: Involvement of 2‐thiouridine tRNA in Signaling Bacterial Sulfur Metabolism
Authors: Black Katherine Ann , Kus Nicole Joanne , Dos Santos Patricia Coutinho 
Published: November 2023

Post‐transcriptional modifications of tRNA are found in all organisms and are critical for their structure and function during translation. 2‐thiouridine (s2U) modification at the wobble position of tRNAGlu, Gln, Lys is required for accurate and efficient protein synthesis and its absence impacts cellular viability. In addition to its role in translation, in yeast, the s2U modification is used as a sensor to gauge nutrient availability and regulate translational capacity and amino acid homeostasis, in which s2U abundance reflects the availability of sulfur‐containing amino acids, cysteine and methionine. In previous work, we showed that B. subtilis s2U biosynthesis involves only two proteins: the cysteine desulfurase YrvO and the thiouridylase MnmA. Genes coding for both proteins are located in an operon directly downstream of the master regulator of cysteine metabolism, cymR, suggesting a link between s2U formation and sulfur amino acid metabolism in this bacterium.In this study we investigated s2U's role in metabolic homeostasis and translational regulation as a sensor of sulfur availability in bacteria. B. subtilis wild‐type was cultured in minimal media containing varying concentrations of an individual sulfur source: sulfate, cysteine, or methionine. Using our established LC‐MS method, analysis of tRNA‐thionucleosides showed that the abundance of s2U correlates to the concentration, but not the source, of sulfur available in the growth media. This relationship appears to be specific to s2U, as the levels of an additional thionucleoside, s4U, remained constant across all conditions; supporting the notion that only s2U is responsive to sulfur availability. Western blot analysis of YrvO and MnmA in these cultures showed patterns consistent with those of CymR expression. That is, higher expression of YrvO and MnmA was observed in cultures using sulfate, cysteine or cystine as the sole sulfur source when compared to thiosulfate, methionine or homocystine. Surprisingly, expression levels of YrvO and MnmA did not correlate with the concentration of sulfur source, or accumulation of s2U thionucleoside, suggesting that the activity of these enzymes is likely controlled by the concentration of cysteine, which affects the catalytic turnover rate of YrvO, the first enzyme in the s2U pathway. In this model, the availability of sulfur controls the flux of sulfur transfer which impacts the overall levels of the signaling metabolite s2U tRNA. Current work is aimed towards evaluating whether s2U levels dictate expression of proteins involved in translation and growth specific processes. Furthermore, these analyses are being expanded to other bacterial species to potentially establish this regulatory role across a diverse group of bacteria.Support or Funding InformationThis work has been funded through the Wake Forest University Center for Molecular Communication and Signaling graduate fellowship.
The Effect of Ultraviolet Stress on Nonfunctional rRNA Decay
Authors: Gomez Karen Janina Villarroel , LaRiviere Frederick J 
Published: November 2023

Nonfunctional Ribosomal RNA Decay (NRD) is a quality control pathway that degrades structurally intact but functionally defective rRNAs. To date, there are two recognized NRD pathways in S. cerevisiae: 18S NRD and 25S NRD. These pathways are responsible for degrading nonfunctional 18S rRNAs and the 25S rRNAs respectively. 18S NRD is translation dependent and involves the translation factor‐like protein Dom34 and its binding partner Hbs1. In contrast, 25S NRD is translation independent and involves the E3 ligase components Rtt101 and Mms1. These pathways were identified using specifically engineered mutant rRNAs, but NRD may normally function to remove accumulated nonfunctional rRNAs that can arise as a result of genotoxic stress such as UV radiation. Understanding rRNA damage and decay is of special importance because rRNA makes up to 90% of the total RNA in S. cerevisiae. Therefore, we are interested in determining the effect of ultraviolet (UV) radiation on NRD to better understand how the 18S and 25S NRD pathways affect cell survival. We hypothesize that by removing damaged rRNAs, NRD can increase cell survival under certain stress conditions. To investigate the effect of UV radiation on NRD function, we transformed BY4741 S. cerevisiae with various rRNA expression plasmids. These plasmids encode either functional or nonfunctional versions of the 18S and 25S rRNAs. The plasmid‐derived rRNAs also contain short, unique sequence tags that allow specific detection of these rRNAs in the presence of total cellular rRNA. The transformed cells were exposed to increasing amounts of UV radiation and cell growth after exposure was monitored in both liquid and solid media. Currently, we are determining relative expression levels of WT and nonfunctional rRNAs using an RT‐qPCR assay. Furthermore, we are continuing these same experiments in NRD‐deficient yeast strains. This work may ultimately help elucidate the role of NRD in cellular recovery from UV damage and contribute to a better understanding of the NRD pathway.Support or Funding InformationThis work is supported by the Howard Hughes Medical Institute.
Development of a Natural Protein Synthesis System from Pseudomonas aeruginosa
Authors: Hughes Casey , Escamilla Yaritza , Hu Yanmei , Bullard James M 
Published: November 2023

We have developed a protein synthesis system from P. aeruginosa composed of the ribosome and 15 accessory proteins. We successfully over‐expressed, purified, and characterized all fifteen of these proteins. These proteins consisted of: PheRS, ArgRS, AspRS, GlnRS, GluRS, LeuRS, MetRS, ProRS, initiation factor‐1 (IF‐1), initiation factor‐2 (IF‐2), initiation factor‐3 (IF‐3), elongation factor Tu (EF‐Tu), elongation factor Ts (EF‐Ts), elongation factor G (EF‐G) and methionyl‐tRNA formyltransferase (MFT). To confirm that the proteins were isolated in active forms, each was subjected to individual activity assays. The aaRS proteins were analyzed using aminoacylation assays. We developed a ribosome dependent GTPase assay to monitor the activity of IF‐2 and EF‐G. EF‐Tu and EF‐Ts activity was monitored by GDP exchange assays. We also devised a ribosome dissociation assay to monitor the activity of IF‐1 and IF‐3. The ability of MFT to formylate Met‐tRNAMet was monitored by measurement of CuSO4 hydrolysis of formylated tRNAMet. We also developed a pseudo‐natural mRNA that allows for monitoring protein synthesis. This mRNA contains high‐usage codons corresponding to tRNA anti‐codons recognized by each of the aaRS proteins present. The assay contains, mRNA, the initiation factors, MFT, elongation factors, ribosomes, aaRS, and amino acids corresponding to each of the aaRS proteins and [3H]Leu. The presence of four codons encoding leucine at the 3′ end of the transcript allows monitoring of full length peptide. Upon completion of synthesis the nascent peptide remains bound to the ribosome and activity is monitored using scintillation proximity assay (SPA) technology. The concentrations of each enzymatic component used in the system was arrived at through sequential rounds of optimization; the concentration of each component represents the concentration just below the saturation point of the titration. This system will be used for screening for chemical compounds that inhibit protein synthesis in P. aeruginosa.Support or Funding InformationResearch was supported by NIH grant 1SC3GM098173‐01A1 and partial student support was from the NIH UTPA RISE program grant # 1R25GM100866‐01 and from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017).
Functional Cooperativity Between the Trigger Factor Chaperone and the ClpXP Degradation System
Authors: Houry Walid A 
Published: November 2023

Comprehensive bioinformatic analyses demonstrated the conservation of the close proximity of the tig gene coding for the ribosome‐associated chaperone trigger factor (TF) chaperone and the genes coding for the ClpXP proteolytic complex suggesting the possible functional association between the protein folding and the protein degradation systems. Genome‐wide genetic interaction studies further supported such an observation. The effect of TF on ClpXP‐dependent degradation varied depending on the nature of the substrate. For some substrates, TF slowed down their degradation by ClpXP, while for others it had no effect. Surprisingly, for a third class of substrates, TF increased the degradation rate. The ClpXP‐dependent degradation of the λ phage replication protein λO was significantly enhanced by TF in vitro and in vivo. TF was found to interact with both ClpX and λO with similar affinities and to enhance the ClpXP‐mediated degradation of ribosome‐stalled λO nascent chains. Experiments suggest that TF transfers λO to ClpX for eventual degradation by the ClpP serine protease and that this could occur cotranslationally. Globally, it is estimated that TF enhances the degradation of about 2% of newly synthesized E. coli proteins. This reveals a new role for TF in facilitating the degradation of newly translated proteins and the potential presence of cotranslational degradation in bacteria.Support or Funding InformationCanadian Institutes of Health Research
Testing Specificity of Commercially Available Nicastrin Antibodies
Authors: Hatter Jessica , Dries Daniel 
Published: November 2023

A protein complex known as γ‐secretase is involved in the development of Alzheimer's disease, which affects cognitive functions such as learning and memory. Nicastrin, a component of γ‐secretase, is the substrate receptor in the protein complex. In a previous research project, the expression of the nicastrin protein was tested by immunofluorescence in a line of nicastrin knockout cells. However, the antibodies stained structures in both wild‐type and knockout cells. This result suggested either of two possibilities: the cells were not nicastrin knockout cells, or the antibodies were nonspecific to nicastrin. Genotyping by polymerase chain reaction (PCR) determined that the nicastrin gene itself was, in fact, knocked out of the cells. To determine if the nicastrin antibodies are specific, western blots were run using the same antibodies with lysates from both wild‐type and knockout cells. By revealing cross‐reactive bands in both lysates, western blots will also aid in determining why the antibodies bind to both knockout and wild‐type cells in immunofluorescence microscopy. Therefore, determining the specificity of commercially available antibodies will better inform both ourselves and the scientific community about using these antibodies for experiments.Support or Funding InformationFunding contributed by the International Healthspan Institute.