bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calsequestrin, a new modulator of unfolded protein response in skeletal and cardiac muscle
Authors: Wang Qian , Groenendyk Jody , Kor Kaylen , Liu Yingjie , Hiess Florian , Chen S.R. Wayne , Knollmann Björn C. , Michalak Marek 
Published: June 2021

Cellular responses to stress are an integral part of muscle physiology and pathology. To deal with stress, cardiac and skeletal muscle cells utilize various mechanisms, engaging intercellular organelles including the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) [1]. The disruptions of ER functions are often observed in ischemic, hypertrophic and failing hearts [1]. However, the molecular mechanisms governing muscle ER stress regulation in the muscle are still unclear. Disruption of ER/SR hemostasis with physiological and pathological stimuli triggers a coping response, so called unfolded protein response (UPR). One of the UPR signaling pathways involves stress sensor inositol‐requiring enzyme 1α (IRE1α), an integral ER/SR membrane protein kinase and mRNA splicing enzyme. Here, we discovered that calsequestrin, a muscle Ca2+ binding protein localized to junctional SR, interacts with UPR stress sensor IRE1α, and contribute to the modulation of stress responses in the muscle. Both skeletal and cardiac isoforms of calsequestrin bind directly to IRE1α as measured by Microscale Thermophoresis (MST), Surface Plasmon Resonance (SPR), and His‐tag Ni‐NTA‐agarose pull down techniques. Although calsequestrin contains three thioredoxin‐like domains its interaction with IRE1α did not involve cysteine residues in IRE1α. Iodixanol gradient fractionation and immunostaining of skeletal muscle sections or isolated cardiomyocytes showed enrichment of IRE1α protein in junctional SR where it is colocalized with calsequestrin. MST analysis revealed that calsequestrin binding to IRE1α has inhibitory effect on IRE1α by preventing dimerization/oligomerization IRE1α required for X‐box binding protein 1 (XBP1) splicing activity. Consequently, cardiac calsequestrin‐deficient cardiomyocytes have increased activity of IRE1α. We concluded that calsequestrin is a new IRE1α binding partner and the protein plays a role in modulation of stress responses in skeletal and cardiac muscle.Support or Funding InformationCanada Institutes of Health Research (CIHR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the binding interactions of AT hook motif variants
Authors: Dobbins Kaitlyn R. , Buchmueller Karen L. 
Published: June 2021

High Mobility Group A (HMGA) proteins are a family of architectural transcription factors that control expression of many genes and their overexpression has been linked to metastasis in numerous cancers. HMGA binds to the minor groove of AT‐rich DNA through three positively‐charged AT hook motifs, each containing the invariant sequence Arg‐Gly‐Arg‐Pro. We endeavor to investigate the effects of changing the amino acids flanking this invariant sequence. Peptides were designed to have varying flanking sequences based on amino acid occurrences in human AT hook proteins. Variations in binding affinities and structural characteristics of these sequences were elucidated using fluorimetry, circular dichroism, and filter binding. Analysis of these variations reveals which amino acids and which amino acid positions in the sequence may be critical to the binding interactions between the AT hook and DNA. These conclusions will inform the development of HMGA‐targeting therapeutics.Support or Funding InformationArnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitophagy during Differentiation of Human Embryonic Stem Cells
Authors: KAO LI‐PIN , Wolvetang Ernst 
Published: June 2021

Mitochondria are cellular organelles that are critical for energy production. They also serve as signalling hubs, integrating a variety of inputs related to metabolic demand, redox control, and apoptosis. The mass and quality of mitochondria within a cell are determined by a balance between mitochondrial biogenesis and mitochondrial turnover. In human embryonic stem cells (hESCs), mitochondrial quality control is especially critical, as this inner‐cell‐mass derived cells are pluripotent and can propagate indefinitely. Cultured hESCs accumulate mutations in the mitochondrial genome, a fact that may limit the use of hESCs (and their differentiated derivate) in regenerative medicine. Mitophagy is a selective form of autophagy in which a mitochondrion is sequestered within a vesicle, delivered to the lysosome, and degraded. Here we show that the number of mitophagic events increases during early stages of hESC differentiation, as indicated by elevated co‐localization between LC3‐GFP‐labeled autophagosomes and MitoTracker‐labeled mitochondria. Furthermore, we detect increased levels of mitophagy in hESCs treated with agents that perturb mitochondrial integrity (e.g., EtBr, which damages mitochondrial DNA, and CCCP, which depolarizes the mitochondrial membrane). Our data demonstrate for the occurrence of mitophagy within hESCs. Our findings further suggest that mitophagy may selectively remove damaged mitochondria as hESCs differentiate. A more thorough understanding of the molecular mechanisms controlling mitophagy in hESCs may eventually allow for manipulation of this process, a technology that could improve the mitochondrial quality of cultured hESCs and thus facilitate the therapeutic application of these cells in regenerative medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dysregulation of Human Mitochondrial ClpP Protease Activity by Acyldepsipeptides Analogs Leads to Apoptotic Cell Death
Authors: Houry Walid A. 
Published: June 2021

Acyldepsipeptides (ADEPs) are potential novel antibiotics. They dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease leading to bacterial cell death. Here, we identified several ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP causing the protease to non‐specifically degrade model substrates. Dysregulation of HsClpP activity by these ADEPs was found to have cytotoxic effects in multiple human cell lines as a result of the activation of the intrinsic, caspase‐dependent apoptosis. ADEP‐HsClpP co‐crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighbouring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a novel therapeutic potential for ADEPs in cancer treatment.Support or Funding InformationThis work was funded by Natural Sciences and Engineering Research Council of Canada grant (RGPIN 2014‐05393) to WAHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4
Authors: Howell Lauren A. , Nemec Antonia A. , Murray Matthew A. , Tomko Robert J. 
Published: June 2021

Turnover of the 26S proteasome by autophagy is an evolutionarily conserved process that governs cellular proteolytic capacity and eliminates inactive particles. In most organisms, proteasomes are located in both the nucleus and cytoplasm. However, the specific autophagy routes for nuclear and cytoplasmic proteasomes are unclear. Here, we investigate the spatial control of autophagic proteasome turnover in budding yeast (Saccharomyces cerevisiae). We found that nitrogen starvation‐induced proteasome autophagy is independent of known nucleophagy pathways, but is compromised when nuclear protein export is blocked. Further, via pharmacological tethering of proteasomes to chromatin or the plasma membrane, we provide evidence that nuclear proteasomes at least partially disassemble before autophagic turnover whereas cytoplasmic proteasomes remain largely intact. A targeted screen of autophagy genes identified a requirement for the conserved sorting nexin Snx4 in the autophagic turnover of proteasomes and several other large multisubunit complexes. We demonstrate that Snx4 cooperates with sorting nexins Snx41 and Snx42 to mediate proteasome turnover, and is required for the formation of cytoplasmic proteasome puncta that accumulate when autophagosome formation is blocked. Together, our results support distinct mechanistic paths in the turnover of nuclear versus cytoplasmic proteasomes, and point to a critical role for Snx4 in cytoplasmic agglomeration of proteasomes en route to autophagic destruction.Support or Funding InformationFlorida State University College of Medicine and NIH/NIGMS grant 1R01GM118600This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Method for Studying Zinc Deficiency in vitro and its Application
Authors: Richardson Christopher Edward , Cunden Lisa Stephanie , Butty Vincent L. , Nolan Elizabeth M. , Lippard Stephen J. , Shoulders Matthew D. 
Published: June 2021

Zinc deficiency is correlated with many pathologies, but the molecular connection between disease states and insufficient zinc is still poorly understood. To investigate the connection between zinc deficiency and organismal phenotypes, we developed a technique to specifically diminish zinc levels in mammalian cell culture media without perturbing the concentrations of other metal ions. Growth of cells in such depleted medium evokes some of the transcriptional responses previously noted in organismal models of zinc deficiency, which can be restored upon addition of zinc to the original levels. RNASeq and miRNA data similarly support a zinc‐specific response to the depletion. We also report how the zinc transcriptome remodels in the context of zinc limiting growth. This robust and transportable methodology offers investigators a unique tool for studying zinc deficiency in the context of mammalian cell culture.Support or Funding InformationThe work was supported by the US National Institutes of Health and the Mallinckrodt Faculty Scholar Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulating ER Protein Folding Homeostasis By Distinctively Processing mRNAs
Authors: Li Weihan , Okreglak Voytek , Peschek Jirka , Kimmig Philipp , Walter Peter 
Published: June 2021

The endoplasmic reticulum (ER) is the major folding compartment for most secretory and plasma membrane proteins in the cell. A conserved signaling pathway, the unfolded protein response, senses and modulates the folding capacity of the ER. To maintain ER protein folding homeostasis under ER stress conditions, the ER membrane embedded sensor, Ire1, initiates two distinct mRNA processing programs through its cytoplasmic kinase/RNase domains. First, in both metazoans and S. cerevisiae, Ire1 catalyzes the unconventional cytoplasmic mRNA splicing of XBP1 (metazoans) or HAC1 (S. cerevisiae)—thereby initiating a transcriptional response that increases the ER folding capacity. Second, in metazoans and S. pombe, Ire1 selectively degrades ER‐localized mRNAs—thereby post‐transcriptionally reducing the ER's protein folding burden through regulated Ire1‐dependent mRNA decay. Thus, Ire1 homologs in S. cerevisiae and S. pombe are specialized to only one of the two functional outputs, while Ire1 in metazoans can perform both. We found that S. cerevisiae and S. pombe Ire1 have distinct RNase specificity, which determine their functional outputs. In addition, we nailed down to three Ire1 residues, which regulate Ire1 RNase specificity by regulating its dimer‐packing. Finally, by applying our new findings, we successfully reconstituted unconventional mRNA splicing in S. pombe cells. Therefore, we engineered S. pombe into a metazoan‐like Ire1 system, where unconventional mRNA splicing and selective mRNA decay co‐exist. Our results provide new insights into a mechanistic understanding of Ire1 function and its interplay with RNA substrates.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DUB activity of Endothelial A20 maintains and repairs endothelial barrier after inflammatory lung injury
Authors: Soni Dheeraj , Wang DongMei , Regmi Sushil C. , Vogel Stephen M. , Tiruppathi Chinnaswamy 
Published: June 2021

Vascular endothelial cadherin (VE‐cad) expression at endothelial adherens junctions (AJs) stabilizes endothelial barrier. Inflammatory mediators induced VE‐cad phosphorylation and ubiquitination promotes its degradation; which in turn destabilizes endothelial barrier to trigger vascular inflammatory responses. However, the intrinsic molecular mechanisms involved in maintaining the basal endothelial barrier stability and the repair of endothelial barrier after vascular injury are poorly understood. Here, we generated endothelial cell (EC)‐restricted Tnfaip3 (A20) knockout (A20ΔEC) mice and showed the pivotal role of the deubiquitinase (DUB) function of A20 in regulating the expression of VE‐cad at AJs. A20ΔEC mice failed to survive sub‐lethal dose of LPS challenge. Consistent with this observation, A20ΔEC mice displayed uncontrolled lung vascular leak and persistent PMN sequestration in lungs in response to LPS. In support of these findings, we observed markedly reduced VE‐cad expression in lungs of A20ΔEC mice. Liposome mediated in vivo delivery of WT‐A20 but not DUB‐inactive (C103A‐A20) mutant construct restored VE‐cad expression in lungs, suppressed LPS induced lung vascular leak and sequestration of PMNs in lungs of A20ΔEC mice. Further, A20 knockdown in human lung microvascular ECs resulted increased VE‐cad phosphorylation on Y685 and Y731, and loss VE‐cad expression at AJs. Forced expression of WT‐A20 but not DUB‐inactive mutant in ECs prevented VE‐cad ubiquitination and degradation. Since induced A20 expression promotes resolution of inflammation, we studied the mechanism of A20 induction in response to TLR4 activation in ECs. Here we observed that IRAK‐M‐mediated NF‐κB activation independent of TAK1 is essential for A20 expression in ECs. Importantly, silencing of IRAK‐M in mouse lung microvessels suppressed expression of A20 and VE‐cad and augmented LPS induced lung vascular inflammatory responses. These findings support that targeting endothelial‐A20 expression is a potential therapeutic strategy to restore endothelial barrier integrity in acute lung injury.Support or Funding InformationNational Institute of Health Grants R01 HL‐128539 and R01 GM‐117028This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Cationic Residue Coordinated to the N1/2O‐Position of FMN in the Nitroreductase Family is Highly Conserved yet not Central to Catalysis
Authors: Musila Jonathan M. , Rokita Steven E. 
Published: June 2021

Enzymes of the nitroreductase structural superfamily form an α+β fold and exist as homodimers with active sites formed at the dimer interface. Although these enzymes contain many conserved residues, their flavin chemistry and catalytic activities are wide‐ranging. For instance, nitroreductase (NfsB) and flavin mononucleotide (FMN) reductases exclusively promote reaction by concurrent transfer of two electrons while iodotyrosine dehalogenase (IYD) and 5,6‐dimethylbenzimidazole synthase (BluB) primarily promote reaction by two single electron transfers. A cationic residue (Arg/Lys) coordinates the N1/2O of the bound FMN throughout the superfamily regardless of their catalytic specificity. To test the necessity of such a highly conserved residue, the candidate residue in IYD (Arg38) was alternatively mutated to a neutral (Gln) and anionic (Glu) derivative. Additionally, the catalytic necessity of an Arg over a Lys was evaluated. The affinity of the R38K and R38Q IYD mutants for iodotyrosine (I‐Tyr) decreased by 11‐ and 46‐fold respectively, compared to that of the wild‐type IYD. In addition, the dehalogenation reaction promoted by R38K and R38Q IYD were suppressed by ~10 and 20‐fold. The most extreme substitution of R38E exhibited no detectable dehalogenation activity but did not prevent FMN from binding to the active site. The R38E IYD also produced a 78‐fold decrease in I‐Tyr affinity.The Arg38 mutations did not, however, alter the FMN chemistry of IYD suggesting that the Arg/Lys at the N1/2O‐position of FMN is not crucial in regulating the one versus two electron transfer process. All variants had comparable FMNox/FMNhq midpoint potentials relative to that of wild‐type IYD. The one electron chemistry of IYD is only expressed in the presence of its substrate or an inert substrate mimic such as fluorotyrosine (F‐Tyr). In the presence of F‐Tyr, the R38Q and R38E variants exhibited an increase of ~50 mV for the midpoint potential of the FMNox/FMNsq couple. In addition, the R38Q and R38E variants registered an increase in the FMNsq/FMNhq midpoint potential of ~50 mV and ~90 mV respectively. Although the R38K IYD had a FMNox/FMNsq midpoint potential comparable to that of the native enzyme, its FMNsq/FMNhq mid‐point potential also increased by ~50 mV. We anticipate similar results with the other model nitroreductases (NfsB and BluB) in this investigation.Support or Funding InformationJHU Provost's Postdoctoral Fellowship and Discovery AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparing E. coli Expression and Enzyme Kinetics of Wildtype and Codon Optimized 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) from Nocardia asteroides A3H1 and Rhodococcus erythropolis IGTS8
Authors: St.George Marissa Louise , Idrizi Keid , Watkins Linette M. 
Published: June 2021

Dibenzothiophene (DBT) and its derivatives comprise up to 60% of the organosulfur contamination of crude oil. The enzyme 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the carbon‐sulfur bond cleavage in the final, and rate‐limiting step in the biodesulfurization of DBT. The wildtype dszb genes from Nocardia asteroides A3H1 (A3H1) and Rhodococcus erythropolis IGTS8 (IGTS8) were expressed with and without chaperone proteins under different induction conditions. Yield of active enzyme was optimal when co‐expressed in E. coli in the presence of GroEL and GroES. Codon optimized dszb genes were synthesized, optimizing E. coli codon usage and minimizing GC content. The codon optimized dszb genes from A3H1 and IGTS8 were expressed with and without chaperone proteins under different induction conditions. GroEL and GroES were both still required for optimal enzyme activity. Protein aggregation was observed using whole cell FT‐IR spectroscopy in all cases, but was significantly larger in induced cells in the absence of chaperones.Support or Funding Information

NSF CHE 1461175
James Madison University Department of Chemistry and Biochemistry
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Natural compounds as probes to dissect lysosome homeostasis
Authors: Li Yang , Hao Xiaojiang , Yang Chonglin 
Published: June 2021

Lysosomes are acidic single‐membrane organelles that serve as the major sites for degradation of cargoes delivered from endocytosis, phagocytosis and autophagy. Recent studies have established lysosomes as important hubs that couple metabolic signaling with cellular clearance. Dysfunction of lysosomes contributes to many human disorders such as lysosome storage diseases (LSDs) and neurodegenerative diseases. To better understand the mechanisms underlying lysosome homeostasis, we screened natural small‐molecule compounds that induce lysosome biogenesis or inhibit lysosome functions. A group of diterpenes, which are activators of protein kinase C (PKC), were found to induce lysosome biogenesis by activating the TFEB transcription factor and inactivating the ZKSCAN3 transcriptional repressor in an mTOR‐independent manner1. In contrast, several tetranortriterpenoids, represented by HLSP, appeared to inhibit the acidification of lysosomes. We found that HLSP also affects intracellular trafficking and autophagy. We are now in the process of investigating the mechanisms of HLSP action by identifying its protein target that is required for generation and maintenance of lysosomal acidity.Support or Funding InformationThis research was supported by grant 21432010 (to X.Hao and C.Yang) from the National Science Foundation of China.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and Synthesis of a Triazole‐based Small Molecule Library for the Inhibition of Bacterial Quorum Sensing
Authors: Kruszynski Catherine E. , Hinspeter Kallie , Danowitz Amy M. 
Published: June 2021

With the rise of antibiotic resistance, finding novel methods of targeting bacteria and treating disease is in high demand. One target system of interest is the bacterial quorum sensing system. This process involves bacteria communicating using small signaling molecules. This communication allows bacteria to engage in group behaviors such as biofilm formation and release of infective agents. It has been demonstrated that synthetic small molecules can be used to inhibit such behaviors. Although some success has been had, the design of more diverse libraries could lead to more efficient modulators, easier synthetic routes, and increased understanding of the quorum sensing pathway. The research presented here focuses on the design and synthesis of a library of adamantyl‐derived triazoles. The triazole scaffold was chosen because of its ease of synthesis, and a wide selection of terminal and di‐substituted alkynes were chosen based on similarities to natural quorum sensing molecules. Some functionalities include, a five membered ring with heteroatoms and carbon chains of varying lengths. Upon synthesis and purification, these molecules are assayed for their ability to inhibit quorum sensing in Vibrio fischeri. Vibrio fischeri is used because its quorum sensing system is similar to other Gram‐negative bacteria that are known to cause human infections (i.e. Pseudomonas aeruginosa), but it is non‐pathogenic. Additionally, this species' bioluminescence is controlled by a quorum‐dependent pathway, which allows for easy detection of quorum sensing inhibition via quantification of light production. To date, the molecules that have been tested have not been found to inhibit bacterial quorum sensing, however, the library continues to grow with different functionalities. Herein, we will describe the design and synthesis of a growing triazole‐based library as well as data regarding the ability of these molecules to inhibit quorum sensing in Vibrio fischeri.Support or Funding InformationCEK received partial funding from the Pennsylvania Academy of Sciences.Mercyhurst University provided the remaining funding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.