bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fine‐Tuning of an Evolutionarily Conserved Histone Chaperone, FACT, by Ubiquitin‐Proteasome System, and its Targeting to the Active Gene by mRNA Capping Machinery to Regulate Transcriptional Elongation
Authors: Kaja Amala , Sen Rwik , Ferdoush Jannatul , Lahudkar Shweta , Barman Priyanka , Bhaumik Sukesh R. 
Published: June 2021

Facilitates Chromatin Transcription (FACT), an evolutionarily conserved histone chaperone involved in multiple cellular processes, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine‐tuned for normal cellular functions. Here, we have used a variety of biochemical, genetic, and molecular biological methodologies to understand regulation of FACT and its abundance. We find that the FACT subunit, Spt16, is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, our results reveal that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similar to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. These results demonstrate for the first time the fine‐tuning of FACT by ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation. Further, it is not clear how FACT is targeted to the active gene. Our recent results reveal that FACT is targeted to the active gene by an mRNA capping enzyme, Cet1, independently of its mRNA capping activity. Subsequently, FACT enhances the release of paused RNA polymerase II from promoter proximal site via Paf1 complex (RNA polymerase II‐associated factor 1 complex) to promote transcriptional elongation. Collectively, these results shed much light on the regulation of FACT and its abundance in controlling transcription, thus providing novel regulatory mechanisms of gene expression.Support or Funding InformationNIH 1R15GM088798‐01 and 2R15GM088798‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression of mTOR Related Molecules in the Placenta of Gestational Diabetes Mellitus (GDM), Intrauterine Growth Restriction (IUGR) and Preeclampsia patients
Authors: Price Kate , Kimbler Brent , Knowlton Nekel , Franson Levi , Hirschi Kelsey M. , Reynolds Paul R. , Arroyo Juan A. 
Published: June 2021

The mechanistic target of rapamycin (mTOR) pathway is involved in placental growth and function during pregnancy. The mTOR pathway responds to nutrient availability and growth factors that regulate protein expression and cell growth. mTOR disruptions are associated with the development of obstetric complications which often result in adverse health outcomes for the mother and/or fetus. The purpose of this study was to identify the differential placental expression of various mTOR‐associated proteins during normal gestation (Control), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Immunohistochemistry was used to stain human placenta for activated proteins (phospho; (p)AKT, (p)ERK, (p)mTOR, (p)p70 and (p)4EBP1. Real‐time PCR array was completed to show differing placental expression of additional mTOR‐associated genes during these conditions. We observed: 1) increased (p)AKT during GDM, 2) increased (p)ERK during IUGR, 3) increased (p)mTOR during GDM and decreased (p)mTOR during IUGR and PE, 4) increased (p)p70 during IUGR and decreased (p)p70 during GDM and PE, 5) increased (p)4EBP1 during GDM, IUGR, and PE, and 6) differential placental expression of mTOR pathway associated genes. We conclude that regulation of the mTOR pathway is uniquely involved in the development of these obstetric complications. These results may provide insight into the physiological relevance of these pathways, and if so, their modification during gestation may help alleviate these diseases.Support or Funding InformationThis work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining a Role for MUC2 Mucin‐Associated Proteins in Colitis and Restitution
Authors: Gorman Hayley , Moreau France , Chadee Kris 
Published: June 2021

Goblet cells produce MUC2 mucin that forms the mucus bilayers in the colon that protect the single layer of epithelial cells lining the gut. The mucus layer serves not only as a viscous physical barrier to protect the epithelial cell surface, but also provides a substrate and nutrient source for colonic microbiota. MUC2 mucin together with microbiota plays a major role in maintaining colonic health and is severely altered in infectious colitis and inflammatory bowel diseases. MUC2 mucin also contains numerous mucus‐associated proteins (MAPs) that are uncharacterized and there is much interest in what role they play in mucosal health and disease. These proteins include calcium‐activated chloride channel regulator 1 (CLCA1), Fc fragment of IgG binding protein (FCGBP) and kallikrein 1 (KLK1). In this study we functionally characterized the expression of MAPs in colonic goblet cells and interrogated their role in colitis and restitution. To quantify MAPs expression we used human LS174T colonic goblet cells and generated CRISPR/Cas9 MUC2 KO cells to determine if MUC2 production regulated the expression of MAPs. Changes in mRNA and protein levels were determined by QPCR and Western blotting, respectively. To model epithelial injury in vitro, LS174T and CRISPR/Cas9 MUC2 KO cells were mechanically wounded. For in vivo studies we used Muc2+/+ and Muc2−/− littermates to basally characterize the expression of MAPs and to quantify what role they play in DSS‐induced colitis. In LS174T cells the temporal expression MUC2, FCGBP, CLCA1 and KLK1 peaked at day 5 and remained high up to day 9, whereas CLCA1 was drastically increased in CRISPR/Cas9 MUC2 KO cells. In LS174T wounded cells, all MAPs increased as early as 2 days post wound that was more pronounced in CRISPR/Cas9 MUC2 KO cells as compared to the LS174T controls. In Muc2+/+and Muc2−/− littermates and organoids derived from Muc2+/+and Muc2−/− colons, MAPs were expressed at similar levels regardless of if Muc2 was present or not. In DSS colitis, MAPs mRNA transcripts and protein levels steadily decreased up to day 15 in Muc2+/+ but not in Muc2−/− littermates. Clca1 expression disappeared during peak of colitis (days 8–12) and Fcgbp, Clca1 and Klk1 were not restored by day 15 during restitution. In active ulcerative colitis (UC), colonic biopsies showed a marked increase in MUC2, FCGBP and KLK1 and a decrease in CLCA1 mRNA expression that returned to basal levels as healthy controls and UC in remission. This study demonstrates that MUC2 mucin and MAPs are not mucus‐associated and are expressed and developmentally regulated independently. In DSS colitis and active UC, MAPs are dysregulated and their reappearance coincides with restitution.Support or Funding InformationSupported by CIHRThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Development and Characterization of IFT20 knockout Mice
Authors: Krump Ryan , Anderson Aleesha , Hietpas Taylor , Pazour Gregory J , Fink Darci M 
Published: June 2021

A primary cilium is a singular cilium on a cell that functions in cell signaling, development and homeostasis. Intraflagellar transport proteins are important for formation of primary cilia and trafficking of proteins to and from the distal tip of the cilium. In mice, tamoxifen‐inducible Cre recombinase‐mediated knockout (KO) of one of these intraflagellar transport proteins, IFT20, results in decreased formation of primary cilia and associated developmental defects. Phenotypic differences, including polydactyly, craniofacial defects, fluid accumulation, and lymphatic defects, were observed between the wildtype and the IFT20 KO, but the exact penetrance of tamoxifen on the inducible Cre‐mechanism that disrupts IFT20 was not known as the induction protocol was relatively conservative. A single dose of tamoxifen was delivered by gavage to pregnant dams at E7 and embryos were harvested and analyzed at E16.5. To infer penetrance of the IFT20 KO, we studied the penetrance of tamoxifen on dual‐reporter mTmG mouse embryos, which followed a similar mechanism as the IFT20 KO model. In this reporter system, cells constitutively expressed membrane‐bound tomato (mT) fluorescent protein; activation of the Cre enzyme in cells exposed to tamoxifen induced expression of green fluorescent protein (mG). Differential expression of mT and mG was assessed by two photon microscopy of whole mount E16.5 embryonic skin. This difference in fluorescence allowed the determination of the penetrance of recombination based on the volume of mG+ cells to the total volume of the tissue sample. The penetrance was quantified manually through ImageJ software. Quantification was difficult due to high background and bright red blood cell autofluorescence in whole mount skin samples. A series of MATLAB‐based clustering and other image analysis methods were used to improve detection of mG+ cells in these tissue samples. In related work, we have identified primary cilia on lymphatic endothelial cells for the first time both in vitro and in vivo and have developed a lymphatic‐specific inducible model of IFT20 disruption.Support or Funding InformationThe funding for this project was provided by the South Dakota State University Department of Chemistry and Biochemistry and BioSNTR of South Dakota.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An allosteric interaction network promotes conformation state‐dependent eviction of the Nas6 assembly chaperone from nascent 26S proteasomes
Authors: Nemec Antonia A. , Peterson Anna K. , Warnock Jennifer L. , Reed Randi G. , Tomko Robert J. 
Published: June 2021

The 26S proteasome is the central ATP‐dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated in part by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation‐specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP‐binding by the ATPase subunit Rpt3 then promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly‐coupled allosteric signals promote chaperone eviction, and provide a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.Support or Funding Information1R01GM118600 to R.J.T.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Ubiquitin‐dependent Switch during Assembly of the Proteasomal ATPases Mediated by Not4 Ubiquitin Ligase
Authors: Park Soyeon , Fu Xinyi , Sokolova Vladyslava 
Published: June 2021

In the proteasome holoenzyme, the hexameric ATPases (Rpt1‐Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early‐stage proteasome assembly, individual Rpt proteins assemble into the hexameric ‘Rpt ring’ through binding to their cognate chaperones: Nas2, Hsm3, Nas6 and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination‐mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step, and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt‐chaperone co‐crystal structures, we show that Not4‐mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring, but hinders incorporation of incoming subunits—Rpn1 ubiquitin receptor and Ubp6 deubiquitinase, thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.Support or Funding InformationBoettcher Webb‐Waring Biomedical Research Award and 1R01GM127688‐01A1 from NIH (S.P.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating
Authors: Tomko Robert J. , Reed Randi G. , Eisele Markus R. , Rudack Till , Schweitzer Andreas , Beck Florian , Nagy Istvan , Pfeifer Guenter , Plitzko Juergen M. , Baumeister Wolfgang , Sakata Eri 
Published: June 2021

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo‐electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution, and report two as‐yet unidentified conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.Support or Funding InformationNIH 1R01GM118600 (RJT) German Science Foundation (Excellence Cluster CIPSM and SFB‐1035/ Project A01) Marie Curie Career Integration Grant (PCIG14‐GA‐2013‐631577) (ES)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant production and structural studies of a serine peptidase from Scopulariopsis koningii
Authors: Pedezzi Rafael , Evangelista Danilo E. , Polikarpov Igor , Cabral Hamilton 
Published: June 2021

Scopulariopsis koningii is an ascomycetes fungus with high biotechnological potential, due to its peptidase. Previous work showed high colagenolytic activity in S. koningii cultured in feather flour rich medium. A native serine peptidase was purified from this bioprocess and has been shown biotechnological potential. However, this fungus has a poor molecular data available in databanks. Thus, we proposed a transcriptomic study to better describing the S. koningii enzymatic arsenal and to find this serine peptidase gene. The gene was in silico selected and synthetized in pPICZα‐A vector to a Pichia pastoris recombinant production. The protein production and purification were carried out in methanol‐induced system followed by Ni‐affinity chromatography. A structural study was carried out by a 3D model construction, generated by homology, and SAXS data collection. The recombinant protein was produced with the pro‐region, which act as an activity inhibitor. The molecular weight of pro‐region plus catalytic region is 40 kDa. SAXS studies showed a globular monomeric protein with Rg ~ 22.5 Å and Dmax ~ 80.3 Å. N‐terminus region build a protuberance related to pro‐region. This region is flexible and, as has been shown elsewhere, acts as activity inhibitor in trans. This structural study has enlightened activation strategies that can bring more control of the peptide hydrolysis start.Support or Funding InformationFAPESP Proc. n° 2016/20385‐2 and 2015/16084‐4 and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the BMP Pathway Type II Receptors in Decidualization
Authors: Hamor Clark , Monsivais Diana , Matzuk Marty 
Published: June 2021

Decidualization is a process driven by hormones and growth factors that results in the differentiation of endometrial stromal cells into decidual cells that are capable of supporting a developing embryo. The overall effect of decidualization is the differentiation of the endometrial stromal cells into decidual cells, which facilitate early blastocyst development, placental function and parturition. Despite the importance of decidualization in reproduction, the molecular mechanisms controlling decidualization are not fully understood. Identifying the signaling pathways responsible for decidualization will provide opportunity to enhance the process of decidualization, which can benefit women experiencing infertility, early pregnancy loss or other pregnancy‐related complications. Past research has shown that Bone Morphogenetic Protein (BMP) signaling is one of the mechanisms controlling decidualization, but the roles of each member of this pathway in decidualization are unknown. Identifying the precise genes in the BMP pathway responsible for decidualization will enable us to develop drugs with clinical applications. The BMP signaling pathway is initiated when a BMP dimer associates with a tetrameric complex of two type one (ALK2, ALK3, ALK6) and two type two receptor proteins (BMPR2, ACVR2A, ACVR2B), which then phosphorylate the SMAD1 and SMAD5 transcription factors. Human endometrial stromal cell (HESC) decidualization can be recapitulated in vitro by exposure to progesterone, estrogen and 8‐bromo‐cyclic‐AMP. Using this technique, we performed siRNA experiments in primary cultures of HESC's to determine the roles of various BMP signaling pathway members in decidualization. We found that SMAD1 and SMAD5 transcription factors and the BMPR2 receptor are essential for in vitro HESC cell decidualization. We also identified that ACVR2A and ACVR2B receptors may be playing a redundant role in decidualization as well. These results indicate that the BMP signaling receptor complex responsible for HESC decidualization is controlled by the concerted action of cell surface receptors that act redundantly but require the activity of both SMAD1 and SMAD5 transcription factors.Support or Funding InformationNCI P30 CURE Grant (CA125123)IRACDA Training GrantPDEP Burroughs Wellcome FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Substrate Binding, Kinetics and Regulation of Glucan Phosphatase Starch EXcess4
Authors: Raththagala Madushi Upendrika , Henao Tiffany 
Published: June 2021

Starch is the main energy reserve in plants, and the activity of dual specificity phosphatase Starch EXcess4 (SEX4) is essential for the degradation of starch via its involvement in the reversible phosphorylation mechanism. In plants, SEX4 acts as the main glucan phosphatase, facilitating the continuous breakdown of the starch granule by amylases. High‐resolution structural studies of SEX4 with the model substrate maltohexaose have provided important insights into the mechanisms of glucan dephosphorylation in plant systems. However, the details of how SEX4 recognizes substrate and binds to the regions of phosphorylated amylopectin within the heterogenous starch granule is not very well understood. Our newly developed Concavalin based assay for determining protein‐carbohydrate affinity increases our understanding of how glucan phosphatases recognize different substrate domains within its microenvironment. Further, enzyme kinetics and the regulatory mechanisms of the SEX4 enzyme are currently not well understood. Here we show the kinetics for SEX4 enzyme using the physiological relevant substrates, amylopectin, and starch. Using these kinetic data, we aim to provide experimental evidence for a possible allosteric regulatory mechanism of SEX4, which may be important for sensing energy status in plants.Support or Funding InformationSkidmore College Startup fundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.