bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distinct Localization of Aurora A Kinase in Mouse Spermatozoa Suggests a Novel Role in Sperm Motility
Authors: Johnson Marquita , Wang Rong , Sperry Ann 
Published: June 2021

In this work, we investigated the interaction of the kinase Aurora A (AURKA) with protein phosphatase 1 (PP1) and its role in sperm flagella. In somatic cells, AURKA and protein phosphatase‐1 (PP1) interact to regulate cilia dynamics and cell division, however a role for this complex in male germ cells had not been shown previously. Primary cilia and flagella are post‐mitotic cellular organelles that extend from the cell membrane and function in signal transduction and motility, respectively. They are analogous structures with both containing a microtubule‐based axoneme. Although the mechanisms governing growth and maintenance of primary cilia are well understood, information regarding the molecular pathways responsible for sperm flagella biogenesis is limited. Based on the role of AURAK and PP1 in cilia and the functional conservation of cilia and flagella, we wanted to determine whether this protein complex plays a role in flagella. Because AURKA is a binding partner of PP1, we investigated whether AURKA interacts with PP1g2, a testis specific isoform of PP1 involved in the initiation of motility. Coimmunoprecipitation experiments provided evidence of an interaction between PP1g2 and AURKA in adult mouse testes. We used indirect immunofluorescent to determine that AURKA localizes to spermatogonia, a finding consistent with its known role in formation of the mitotic spindle. We also detected AURKA in the flagella of spermatids within the lumen of seminiferous tubules prior to spermiation. Most notably, AURKA was localized to the flagella of spermatozoa. AURKA localized to the principal piece of epididymal sperm while activated AURKA (phospo‐Th288) localized to the midpiece of sperm flagellum. In order to determine whether the activity of AURKA changed during maturation of sperm in the epididymis, we quantified the activation of AURKA in sperm collected from the caput, corpus and cauda epididymis. Activated AURKA is enriched in the cauda epididymis where motile sperm reside. Results obtained from this study suggest a novel role for AURKA in the signaling pathway controlling sperm motility through an interaction with PP1.Support or Funding InformationThe work is supported by grant HD08051 Eunice Kennedy Shriver National Institute for Chile Health and Human DevelopmentThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RecQ4 helicases stimulate nuclease activity during DNA inter‐strand crosslink repair.
Authors: Bochman Matthew L. , Rogers Cody M. 
Published: June 2021

Mutations in the RecQ4 DNA helicase cause genomic instability that leads to a predisposition to various cancers and/or premature aging. Despite its importance to human health, however, RecQ4 remains the least studied of the five human RecQ helicases because it is difficult to work with both in vivo and in vitro. We recently characterized a putative RecQ4 homolog from Saccharomyces cerevisiae called Hrq1 and found that like RecQ4, it is involved in telomere maintenance and DNA inter‐strand crosslink (ICL) repair. Our genetic, biochemical, and structural data indicate that Hrq1 is a bona fide RecQ4 homolog and represents a simple model to study the roles of RecQ4 in genome integrity. Currently, we are focusing on Hrq1's mechanism of action during ICL repair. S. cerevisiae cells lacking the HRQ1 gene or expressing a catalytically inactive allele (hrq1‐K318A) are highly sensitive to the ICL‐inducing drugs such as mitomycin C, and this sensitivity is epistatic to mutations in the nuclease Pso2, which defines the major ICL repair pathway in yeast. In contrast, Hrq1 does not function in the proto‐Fanconi Anemia pathway in S. cerevisiae. By analogy to another well characterized RecQ helicase/nuclease pair in yeast that is responsible for end resection during double‐strand break repair (Sgs1/Dna2), we hypothesized that Hrq1 may physically interact with Pso2 to facilitate ICL repair. Using purified proteins, we found that Hrq1 stimulates Pso2 nuclease activity on short DNA substrates. Further, reciprocal pull‐down experiments indicate that Hrq1 and Pso2 physically associate, suggesting that nuclease stimulation is direct rather than the result of Hrq1 generating a preferred substrate for Pso2. Similar in vitro nuclease stimulation is achieved with recombinant RecQ4 but not with S. cerevisiae Sgs1, suggesting that it is specific to RecQ4 family helicases. Experiments are now underway using bacterial and archaeal RecQ4 homologs to determine if this interaction is evolutionarily conserved among all domains of life and to identify the interaction motifs on the proteins.Support or Funding InformationSupport was provided by the College of Arts and Sciences, Indiana University, an Indiana University Collaborative Research Grant from the Office of the Vice President for Research, and the American Cancer Society [RSG‐16‐180‐01‐DMC].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Set4 Promotes Survival during Oxidative Stress through Regulation of Stress Response Genes
Authors: Tran Khoa , Jethmalani Yogita , Jaiswal Deepika , Green Erin M. 
Published: June 2021

The yeast protein Set4 contains both a PHD finger and a SET domain, a common signature of chromatin regulatory proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET and the human protein MLL5. Although MLL5 has been implicated in diverse pathways such as hematopoiesis and spermatogenesis, as well as disease states including neurodevelopmental disorders and cancer, its cellular function is still largely unclear. The biological role for Set4 and its function in chromatin regulation has not been investigated. Set4 had previously been reported to be lowly expressed under normal growth conditions, and therefore not expected to have a substantial role in chromatin function or gene expression regulation. However, our data indicated that it plays a highly‐specialized role during stress responses, and that it is a key context‐dependent chromatin regulator. Specifically, we have determined that Set4 activity is critically‐balanced in the cell to promote survival specifically during oxidative stress. Gene expression analysis indicates that the role for Set4 during oxidative stress is to activate genes that promote survival in the presence of oxidative insults. Our data further indicate that Set4 interacts with chromatin in a highly‐regulated manner, and it directly associates with stress‐response genes upon oxidative damage. Our results suggest a model in which Set4 is a stress‐responsive chromatin protein that promotes the activation of gene expression programs required for cellular protection during oxidative stress. To our knowledge, this work is the first to define a biological role for Set4, a conserved SET‐domain protein in yeast that was predicted, although not yet characterized, to be a chromatin regulator. These results further our understanding of mechanisms that protect cells during oxidative stress, and will open new avenues for the investigation of the Set3‐Set4 subfamily of SET‐domain containing proteins and their roles in gene expression control in response to fluctuating environmental conditions.Support or Funding InformationNIH R01GM124342This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histone chaperone Nap1 facilitates histone dynamics in the nucleosome
Authors: Lee Tae‐Hee , Lee Jaehyoun 
Published: June 2021

We developed a single‐molecule FRET (smFRET) experimental system with which we can separately monitor histone H2A‐H2B dimer dynamics and DNA dynamics within a nucleosome particle. Utilizing the system, we monitored the nucleosome dynamics in the presence of histone chaperone Nap1 in order to investigate how the chaperone alters the dynamics of the nucleosome with and without histone acetylations at H3K56 and H4K16. We also compared the nucleosome dynamics in the presence of Nap1 to that in its absence at an elevated salt concentration. Our results indicate that DNA unwraps out of the nucleosome before histone dimer moves at an elevated salt concentration of 650 mM NaCl. On the contrary, we observe over 60 % of the nucleosomes show dimer motion prior to DNA unwrapping in the presence of Nap1. Upon monitoring the DNA unwrapping kinetics with histone acetylation at H3K56 or H4K16, we found that DNA unwrapping becomes facilitated mainly by destabilizing the fully wrapped nucleosomal state. Our results strongly suggest that Nap1 would have a direct impact on RNA polymerase II progression through the nucleosome, which we have also confirmed with our system, and that histone acetylations would amplify this effect in the presence of Nap1.Support or Funding InformationNIH [GM097286]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1000 Ways to Die: Synthetic Lethality with an HDAC
Authors: Alexandre Karlbuto , Hodges Evan , Ciccaglione Kerri , Law Michael 
Published: June 2021

Histones are proteins that can be post‐translationally modified and these modifications will determine the expression of genes. Many studies have shown that it is very common for cancers to have mutations in their histone genes. The concept of synthetic lethality states that if a cell has a single mutation it will be able to survive. But if a second mutation is applied, the cell will become synthetically sick and/or die. In our study, we are seeking to determine synthetically lethal interactions between the histone H3 gene and RPD3 histone deacetylase enzyme. In order to discover potential genetic targets to develop therapies against cancers. To achieve this we used a genetically engineered yeast strain that has the histone H3 gene and RPD3 gene deleted. The H3 gene deletion is covered by a wild type H3 plasmid. These yeast were then transformed with plasmids from the SHIMA library. The yeast were then grown on plates lacking uracil and tryptophan. The individual colonies were then patched and replica plated onto tryptophan deficient media. Finally, they were replica plated onto agar plate media containing 5‐ FOA. The DNA from yeast deemed synthetically lethal or sick were then put through “Library Prep” which is a high throughput method used to isolate the H3 gene within the plasmid with low error rate. The lethals were then put into Next Generation sequencing, aligned, and transferred in the IGV. Finally, we were able to estimate with high accuracy the frequency of H3 mutants in our lethal pool. The top three frequencies discovered were F67A, F84A, and Q93A. They represented 35%, 12% and 10% of our lethal pool respectively. In total we discovered ten new potential synthetically lethal interactions between histone H3 and RPD3. We also used this information in combination with previous knowledge to generate a genetic interaction map for RPD3 from our findings. Although still in the preliminary stages when verified with duplication studies our new potential synthetically lethal interactions can be targets to develop cancer therapies. Also, the knowledge gained from our study can be used to understand genetic interaction between histones and histone modification enzymes.Support or Funding InformationSummer Medical Research Fellowship at Rowan University School of Osteopathic MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis for H2B Monoubiquitylation by Bre1 and Rad6
Authors: Hyun Kwangbeom , Su Xiaonan , Li Haitao , Kim Jaehoon 
Published: June 2021

Histone ubiquitylation is implicated in many cellular processes that include transcriptional regulation and DNA damage repair. In yeast, monoubiquitylation on lysine 123 residue of histone H2B is mediated by Rad6 E2 ubiquitin conjugating enzyme and Bre1 E3 ubiquitin ligase. Interestingly, our biochemical analyses previously showed that Rad6 binds to the N‐terminal coiled‐coil motif (CCM) of Bre1 while most E2 enzymes interact with RING‐finger domains within their cognate E3 enzymes. To understand detailed structural basis for Rad6‐Bre1 interaction, we solved the crystal structure of Rad6‐Bre1 CCM complex at 2.1Å resolution and deployed in vitro protein interaction and H2B ubiquitylation analyses with purified factors to validate obtained structural model. We found that Bre1 CCM anchors to the backside of Rad6 and this unique Rad6‐Bre1 recognition mode directs histone H2B monoubiquitylation rather than polyubiquitylation. In addition, two Bre1 CCM monomers form a homodimer in an asymmetric manner with each adopting a distinct conformation. This explains why two Bre1 homologs, Bre1A and Bre1B, are evolved to form a heterodimeric E3 enzyme in higher eukaryotes. Our structural study reveals a novel RING‐finger independent interaction of Rad6 and Bre1 and establishes the molecular mechanism of H2B monoubiquitylation.Support or Funding InformationNational Research Foundation (NRF) of Korea (2015R1A1A1A05001593)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of SOCS5 as a gene target of miR‐9 in inflammatory monocytes
Authors: Stander Matthew , Rabquer Bradley 
Published: June 2021

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammatory joint destruction. The inflammation is caused in part by monocyte migration into the joints and subsequent macrophage differentiation. MicroRNA (miRNA) are short, single‐stranded, noncoding RNA that bind to mRNA to regulate gene expression. Our lab has previously found that miR‐9 was upregulated in monocytes isolated from patients with RA. SOCS5 is part of the suppressor of cytokine signaling (SOCS) family which downregulates cytokine expression such as epidermal growth factor receptor and interleukin‐6. We hypothesize that SOCS5 is a gene target of miR‐9 and propose a luciferase assay to confirm binding.Using microRNA.org, SOCS5 was determined as a target gene of miR‐9 in silico with a mirSVR predicted binding score of −2.17. microRNA.org uses a combination of miRanda, an algorithm to predict miRNA binding sites, and mirSVR, a support vector regression that provides a predicted binding score. PCR primers that amplify the miR‐9 3′ UTR binding site of SOCS5 were designed using primer‐BLAST. U937 monocytes were grown, mRNA was isolated, and cDNA was produced. The PCR product and pmirGLO vector were digested with Sac1 and Sal1 restriction enzymes and then ligated. The ligated vector was transformed into E. coli cells. Plasmid vector was isolated from single colonies. We determined the ligation and transformation were successful by performing PCR using sequencing primers that amplify the multiple cloning site. Gel electrophoresis was used to separate and view the ligated plasmid PCR product. The SOCS5 insert was calculated to be 576 bp, whereas the non‐ligated plasmid came out to be 380 bp. The ligated plasmid was sequenced to confirm ligation.In conclusion, we have determined that SOCS5 is expressed by U937 monocytes. We have also successfully cloned the 3′ UTR miR‐9 binding site of SOCS5 into the pmirGLO vector for use in luciferase assays to assess binding between miR‐9 and SOCS5.Support or Funding InformationAlbion College Biology Department and The Foundation for Undergraduate Research, Scholarship, and Creative Activity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection and comparison of circular RNAs in mouse striatum and retina
Authors: Chen Jian‐Huan 
Published: June 2021

PurposeCircular RNAs (circRNAs) are a type of non‐coding RNAs (ncRNAs), and could be potential clinical biomarkers in complex disorders of the nervous system. Expression of circRNAs possess spatio‐temporal properties. Herein we used RNA sequencing (RNA‐Seq) to detect and compare circRNAs and their expression pattern in the mouse striatum and retina.MethodsWhole striatum and retina was collected from adult mice, and total RNA was extracted using TRIzol followed by phenol/chloroform processing. RNA‐Seq libraries were prepared from total RNA using the ribosomal RNA depletion method. RNA‐Seq was performed for three striatum and three retina samples on the Illumina HiSeq X10 platform with a configuration of 90 million 150 bp pair‐end reads per sample. Reads was aligned to the mouse full genome (UCSC version mm10) and splice junctions were identified by using RNA STAR version 2.5. circRNAs were detected and quantified using DCC version 0.4.4, and difference of circRNA expression between the striatum and retina was analyzed using CircTest. Functional analysis including gene ontology (GO) enrichment and KEGG pathway was performed using DAVID bioinformatics resources 6.8.ResultsIn total 781 circRNAs were detected and passed quality control in the striatum and retina, among which 342 showed significant expression difference between the two types of neural tissue. Among the differentially expressed circRNAs, 5 were only detected in the striatum and 29 only in the retina. Functional Annotation Clustering analysis using DAVID showed that a total of 70 host genes of differentially expressed circRNAs were enriched in K48‐linked ubiquitination and positive regulation of cell cycle, and 39 of these genes had a high expression level in the brain.ConclusionIn the current study, our results demonstrated remarkable difference in circRNA patterns between two types of neural tissue including the striatum and retina, suggesting the potential importance of circRNAs in development and differentiation of nerve system.Support or Funding InformationThe Natural Science Foundation of China (No. 31671311, 81371033 and 81000397), the “Six Talent Peak” Project of Jiangsu Province (SWYY‐127), the Guandong High‐level Personnel of Special Support Program, the Yangfan Plan of Talents, and the Fundamental Research Funds for the Central Universities (JUSRP51712B).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LncRNA GAS8‐AS1 directs epigenetic modulation and activation of the tumor suppressor GAS8 via MLL1/WDR5 to suppress hepatocarcinogenesis
Authors: Yang Ming 
Published: June 2021

Long noncoding RNAs (lncRNAs) are vital players in hepatocellular carcinoma (HCC). We previously identified a significantly mutated lncRNA GAS8‐AS1 locating in intron 2 of GAS8. However, its involvement in HCC is still largely unknown. In this study, we thoroughly functional evaluated its critical role in regulating chromatin dynamics, gene expression, cancer development and sorafenib multi‐kinase target therapy in HCC. We for the first time declare that GAS8‐AS1 and its host gene GAS8 act as novel HCC tumor suppressors employing a series of in vitro and in vivo assays. Consistent with this notion, a significantly decreased GAS8‐AS1 or GAS8 expression exists in HCC tissues and is associated with poor prognosis of HCC patients. Interestingly, lncRNA GAS8‐AS1 could evidently promote GAS8 transcription. Although GAS8‐ AS1 does not impact promoter DNA methylation of GAS8, we successfully annotated two lncRNA interacting proteins, Mixed Lineage Leukemia 1 (MLL1), a histone 3 Lys4 (H3K4) methyltransferase, and its partner WD‐40 Repeat Protein 5 (WDR5) based on bioinformatics methodology. Chromatin immunoprecipitation and RNA immunoprecipitation assays elucidate that lncRNA GAS8‐AS1 is required to maintain the GAS8 promoter in an open chromatin state. In accord with this, knockdown of GAS8‐AS1 results in reduced MLL1/WDR5 binding, decreased H3K4me3 levels, diminished RNA Pol II functions and gene silencing of GAS8. GAS8‐AS1 may function as part of a surveillance mechanism that keep activation of GAS8 promoter and transcription, which thus prevents hepatocarcinogenesis. Our results highlight the prevalent involvement of regulatory lncRNAs in cancers and provide pathogenic insights into HCC development and treatment.Support or Funding InformationNational Natural Science Foundation of China (31671300); Taishan Scholars Program of Shandong Province (tsqn20161060)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutation W209R in Human D‐amino acid oxidase protein
Authors: Birdsong Garrick Leif , Jonsson Amanda L. 
Published: June 2021

Human D‐amino acid oxidase (hDAAO) is an enzyme found in the brain that is responsible for breaking down the signaling molecule D‐serine. hDAAO is composed of two identical subunits that each bind FAD as a cofactor. Increases in hDAAO activity will lead to too much hydrogen peroxide being produced as a product and a decrease in activity will lead to high levels of D‐serine. Because of this, mutations in hDAAO are linked to several disorders such as schizophrenia or amyotrophic lateral sclerosis. One particular mutation involves residue 209, which is located at the dimer interface, far from FAD and the active site. In the wild‐type, residue 209 is a tryptophan and in the mutant it has changed to an arginine; this W209R mutation increases the activity of the enzyme. Molecular dynamics simulations were used to analyze how the protein structure changes over time for both the wild‐type and W209R mutant hDAAO enzyme to help determine how changing a single residue far from the active site can impact protein function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteasomal ATPases Hard at Work: The Inner Workings of a Protein Destruction Machine
Authors: Snoberger Aaron M. , Smith David M. 
Published: June 2021

Across all domains of life, the proteasome is responsible for the majority of targeted protein degradation in the cell. Despite its crucial role in virtually every cellular process, our understanding of how the proteasome operates from a mechanistic perspective is still highly limited. The proteasomal ATPases are the epicenter of the 26S proteasome, as they constitute the primary machinery that catalyze the rate limiting step in substrate degradation: Protein translocation into the proteolytic particle. In order to understand how these complex molecular motors function, we study the archaeal proteasomal ATPases (PAN). In both the 26S ATPases and PAN, we have previously shown that energy from ATP hydrolysis is tightly coupled to unfolding, and we have proposed that such tight regulation is the result of highly coordinated ATP binding and hydrolysis. We could determine the stoichiometry of ATP binding (2 high and 2 low affinity sites per hexamer) as well as their position relative to one another (neighboring subunits), but since PAN is a homohexamer we were unable to distinguish which subunits were responsible for ATP binding. In the present study, we utilized an extensive disulfide crosslinking approach to determine that PAN's N‐domains exist in distinct conformations that we were able to capture. This allowed us to correlate specific N‐terminal conformations with functional activities with minimal structural perturbations. This approach is also allowing us to correlate N‐terminal conformations with substrate recruitment and regulation of ATP‐binding. These studies further provide direct evidence to support our previously reported trans‐arginine finger “sensing” model that describes how the ATPases translocate protein substrates.Support or Funding InformationNIH‐R01GM107129 F31GM115171This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.