bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli
Authors: Chng Shu‐Sin , Yeow Jiang , Tan Kang Wei , Holdbrook Daniel , Chong Zhi‐Soon , Marzinek Jan , Bond Peter 
Published: June 2021

A distinctive feature of the Gram‐negative bacterial cell envelope is the presence of an asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, thus allowing survival of bacteria in harsh environments. In Escherichia coli, the OmpC‐Mla system is responsible for maintenance of OM lipid asymmetry. Osmoporin OmpC and the OM lipoprotein MlaA form a complex proposed to remove PLs from the outer leaflet of the OM. How this complex is organized in the OM to perform this function is not known. In this report, we define the molecular architecture of the OmpC‐MlaA complex to gain insights into its function in PL transport. We show that MlaA sits entirely within the OM lipid bilayer, and interacts with the OmpC trimer at its dimeric interfaces. Molecular dynamics simulations reveal a membrane‐spanning hydrophilic channel within MlaA, suggesting a path for PL translocation across the OM. We demonstrate that a hydrophobic hairpin loop adjacent to this putative channel is critical for modulating the activity of MlaA; restricting flexibility of this structure significantly perturbs the function of the complex. Finally, we establish that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work provides a glimpse into the molecular mechanism of how the OmpC‐MlaA complex may extract PLs from the outer leaflet of the OM, and highlights key features that could be exploited in the development of future antimicrobial drugs.Support or Funding InformationNational University of Singapore Graduate School for Integrative Sciences and Engineering scholarship (to J.Y.).Singapore Ministry of Education Academic Research Fund Tier 3 grant (MOE2012‐T3‐1‐008) grant to (P.J.B.).Singapore Ministry of Education Academic Research Fund Tier 1 and Tier 2 (MOE2013‐T2‐1‐148) grants (to S.‐S.C.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Unfolded Protein Response Regulator, ATF6, Promotes Mesodermal Differentiation
Authors: Kroeger Heike , Grimsey Neil , Paxman Ryan J. , Chiang Wei‐Chieh , Plate Lars , Jones Ying , Shaw Peter X. , Trejo JoAnn , Tsang Stephen H. , Powers Evan , Kelly Jeffery W. , Wiseman R. Luke , Lin Jonathan H. 
Published: June 2021

ATF6 encodes a transcription factor that is activated during the Unfolded Protein Response to protect cells from ER stress. Loss of ATF6α and its paralog ATF6β, results in embryonic lethality, notochord dysgenesis, and in people, loss of ATF6α specifically, results in malformed neuroretina and congenital vision loss. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated the function of ATF6 in development using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a recently identified small molecule ATF6 agonist, and we inhibited ATF6 using iPSCs from patients harboring ATF6 mutations. We discovered that ATF6 suppresses pluripotency, enhances differentiation, and surprisingly, guides stem cells toward mesodermal cell fates. Our findings reveal a novel role for ATF6 during differentiation and identify a new strategy to robustly create mesodermal tissues through modulation of the ATF6 arm of the UPR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential effect of OCRL1 patient mutations on protein localization, and sensitivity to FDA‐approved drug suppression of cellular phenotypes associated with Lowe syndrome
Authors: Ramadesikan Swetha , Madhivanan Kayalvizhi , Aguilar R. Claudio 
Published: June 2021

Lowe Syndrome (LS) is a lethal X‐linked genetic disorder caused by mutations in the OCRL1 gene, which encodes the lipid phosphatase Ocrl1. LS children present with congenital cataracts, low molecular weight proteinuria and mental retardation as characteristic clinical symptoms. Progressive renal dysfunction is the main cause of death. Ocrl1 localizes to the trans Golgi network (TGN), early endosomes and the plasma membrane and has been shown to participate in vesicle trafficking. We established that LS patient cells are defective in fluid phase uptake and ciliogenesis, which are critical processes required for kidney function. Over 100 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. However, our results indicate that different mutations have diverse effect on Ocrl1 localization and on triggering cellular phenotypes.We expressed GFP‐Ocrl1 (WT and several patient mutants) in human kidney proximal tubule (HK2) cells and LS fibroblasts lacking endogenous Ocrl1. The co‐localization of WT/mutant Ocrl1 with various organelle markers was quantitatively analyzed and different cellular processes/phenotypes were monitored. Our data indicates that in general, N‐terminal PH domain mutations resulted in redistribution of Ocrl1 to the cytoplasm, ASH‐RhoGAP domain mutations prevented localization of Ocrl1 to TGN and mutations in the phosphatase domain resulted in Golgi apparatus (GA) morphological abnormalities.Importantly, using a drug repurposing approach we identified two FDA‐approved compounds able to differentially suppress LS cellular phenotypes; the ability of these drugs to alleviate defects associated with specific Ocrl1 patient mutants was also analyzed. Our data indicate that while some patient OCRL1 mutations require treatment with both drugs, others only necessitate one for LS cellular phenotype suppression.This study determined for the first time the effect of different patient mutation on the Ocrl1 intracellular localization and on various LS‐associated cellular phenotypes. Further, it explored how different OCRL1 mutations determined the sensitivity of cells to the newly identified FDA‐approved drugs with ameliorating effects on LS phenotypes. It also contributes to expand our understanding of the molecular mechanism of LS pathogenesis and provide the basis for the development of a LS‐specific therapeutic.Support or Funding InformationLowe Syndrome TrustNational Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Metabolism and Mitochondrial Lipid Content Drive the Evasion of Apoptosis
Authors: Ross Kyle A. , Wyeth Alexandra J. , Dittenhafer‐Reed Kristin E. 
Published: June 2021

Mitochondria are the metabolic centers of the cell and critical players in initiating programmed cell death. Mitochondrial dysfunction and dysregulated apoptosis contribute to a number of disease states, including inherited mitochondrial syndromes, neurological disorders, and cancer. Critical to both mitochondrial function and apoptosis is the architecture of mitochondrial membranes. A mitochondrion is comprised of an inner and outer membrane composed of numerous phospholipids, including cardiolipin, a phospholipid found exclusively in the mitochondria. Cardiolipin binds to cytochrome c in the inner mitochondrial membrane, preventing its release and the initiation of intrinsic apoptosis. Defects in genes required for the maintenance of mitochondrial membrane structure and cardiolipin composition have been identified, and represent an emerging class of mitochondrial disorders. However, a detailed molecular understanding of the synthesis and regulation of mitochondrial membrane phospholipids is lacking. The overall objective is to investigate mechanisms of increased cardiolipin biosynthesis and to study the role of cardiolipin content and composition in regulating the initiation of intrinsic apoptosis. Cancer cells are often characterized by an increased dependence on glucose. These cancers are often resistant to chemotherapies, representing a significant clinical problem. Current research employing cellular phospholipid quantitation has identified that altered cellular glucose metabolism regulates cardiolipin synthesis. Further, the effects of altered cardiolipin levels on cell viability and cytochrome c release upon RNA mediated knockdown of cardiolipin biosynthesis enzymes were studied. Data acquired from cell viability and immunofluorescence microscopy assays suggest that knockdown of cardiolipin synthase (CRLS1), resulting in decreased cardiolipin levels, decreased cell viability after drug treatment, and increased cytochrome c release. Alternatively, loss of lysophosphatidic acid phosphatase type 6 (ACP6), hypothesized to increase cardiolipin levels, resulted in decreased cytochrome c release, which increased cell viability. These results will allow for an increased fundamental understanding of cardiolipin physiology in order to uncover the therapeutic potential of targeting new mitochondrial pathways to treat human disease like cancer.Support or Funding InformationDow Scholars ProgramHope College Division of Natural and Applied SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protease OMA1 modulates mitochondrial metabolism and cristae structure through interaction with MICOS complex
Authors: Levytskyy Roman Myroslavovych , Viana Martonio Ponte , Khalimonchuk Oleh 
Published: June 2021

Mitochondria are highly dynamic organelles, whose metabolic outputs are known to correlate with changes in the organelle's network and ultrastructure. Defects in these activities appear to underlie a variety of human diseases, however many molecular aspects of mitochondrial involvement in these pathologies are far from clear. Mitochondrial inner membrane protease OMA1 is a critical regulator of mitochondrial dynamics in higher eukaryotes. It is best known to modulate mitochondrial morphology through proteolytic processing of the dynamin‐like GTPase OPA1, thereby promoting rapid partitioning of mitochondrial network in response to various homeostatic challenges. OMA1 is also known to perform protein quality control functions, and to maintain the stability of respiratory supercomplexes, thus influencing mitochondrial energy metabolism. Mechanistic aspects, as well as functional connection of these important activities remain largely unaddressed.Here we demonstrate that in addition to its role in mitochondrial fragmentation, OMA1 is required to maintain mitochondrial ultrastructure through an interaction with mitochondrial contact site and cristae organizing system (MICOS) ‐ a protein complex critical for stability of mitochondrial architecture. This specific interaction is mediated by MIC60 MICOS subunit, and – under conditions of cellular stress – influences the recruitment of MICOS proteins MIC10 and MIC27 without affecting mitochondrial fission and fusion machinery proteins. We further show that OMA1‐MICOS association is important for metabolic fine‐tuning of mitochondria in response to homeostatic insults such as mitochondrial depolarization. Moreover, in the absence of OMA1, these activities can be partially compensated for by expression of an artificial tether protein that bridges outer and inner mitochondrial membranes, thereby emulating tunable MICOS complex.Our findings demonstrate that OMA1‐mediated support of mitochondrial ultrastructure is important for modulation of mitochondrial bioenergetic outputs in response to homeostatic challenges and highlight previously unanticipated role for this protease in mitochondrial physiology.Support or Funding InformationNational Institutes of Health grants R01GM108975 (to O.K.) and P30 GM103335 (to Redox Biology Center at UNL)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 O‐GlcNAc Hydrolase with Phosphomimetic Mutations
Authors: Hinkle Megan , Crawford Garland 
Published: June 2021

O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in post‐translational modification. This enzyme hydrolyzes a single O‐GlcNAc from serine or threonine residues on target proteins located in the cytosol of metazoan organisms. OGA is the only protein known to carry out this function in the context of cytosolic proteins. This exclusivity of function makes OGA a likely target of post‐translational modifications in order to regulate its activity. Our aim is to generate a phosphomimetic OGA with altered enzymatic activity by using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid. The end goal of this research being to introduce the mutated OGA gene into a mammalian cell system to study the relationship between OGA phosphorylation and its activity. Analysis by Western blot will reveal the extent to which the mutants alter OGA expression levels and O‐GlcNAc modification levels in the cell. Additionally, interactions of OGA with known binding partners, such as OGT, the enzyme which adds the O‐GlcNAc sugar to target proteins, can be studied with co‐immunoprecipitation. This work lays the foundation for identifying potential phosphorylation states of OGA and would aid in elucidating the regulatory pathways of this enzyme.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening Point Mutations of the O‐GlcNAc Hydrolase Enzyme, OGA, to Investigate Potential Regulation
Authors: Crawford Garland 
Published: June 2021

The O‐GlcNAcase enzyme (OGA) hydrolyzes the glycosidic linkage between a GlcNAc and the hydroxyl group on serine and threonine residues on target proteins. OGA is the sole enzyme responsible for removing this cytosolic, post‐translational modification which makes the enzyme itself a likely target for post‐translation modification. Several groups using proteomic analysis have identified phosphorylation sites on OGA, but the functional consequences of these changes have not been fully investigated. In an effort to characterize potential sites of regulation, a plasmid containing 6xHis‐OGA was altered by site‐directed mutagenesis to produce phosphomimetics by substituting an aspartic acid or glutamic acid for a serine or threonine. These mutant OGA enzymes were expressed in E.coli and purified using a two‐step purification of Ni2+‐metal affinity followed by size exclusion. Enzyme activities were investigated using a paranitrophenol‐GlcNAc substrate and were compared to the wild‐type enzyme. The goal of this research is to better understand the function and regulation of OGA, and the role that this important enzyme plays in signaling cascades.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Pancreatic Islet Microtissue 3D Characterization for Compound Screening
Authors: Villani Thomas , Johnson Michael , Gardner Graeme 
Published: June 2021

In the last few years several companies have developed 3D in vitro cell culture models and have demonstrated that these 3D models more accurately depict in vivo characteristics compared to traditional 2D monolayer cell culture. Because of this improved in vivo relevancy, these models are being rapidly adopted in the drug discovery space as they can more accurately predict in vivo efficacy and toxicological characteristics. Of the different types of 3D cell culture models being used today, pancreatic islet microtissues have been shown to be a powerful tool for assessing the effectiveness of compounds on diabetes. However, current imaging approaches (e.g. confocal microscopy, wide‐field microscopy) are limited in their ability to completely characterize these 3D models as light attenuation limits imaging depth to approx. 1–3 cell layers. Therefore, current imaging techniques are not able to completely characterize the effect of compounds on beta cell proliferation. Through this work, pancreatic islet microtissues of approx. 250 μm diameter were exposed to several compounds that induce beta cell proliferation and the microtissues were then stained for EdU or Ki67, glucagon, insulin and DAPI. The labeled microtissues were then rendered transparent with a rapid and plate‐compatible tissue clearing technique that allows for the microtissues to be imaged in their entirety using high content confocal imaging. It was shown through this work that the addition of a tissue clearing technique resulted in a 3‐fold increase in the number of cells characterized in the microtissues compared to traditional wide‐field or confocal microscopy.Support or Funding InformationThis work was support by Visikol Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Highly efficient long‐acting cocaine hydrolases as a treatment for cocaine addiction
Authors: Zhan Chang‐Guo , Zheng Fang 
Published: June 2021

AimsEnzyme therapy using a highly efficient exogenous cocaine‐metabolizing enzyme is recognized as a promising new therapeutic strategy for cocaine abuse. Our recently designed highly efficient, long‐acting cocaine hydrolases (LA‐CocHs), engineered from human butyrylcholinesterase, have a considerably improved catalytic efficiency against cocaine and a significantly improved biological half‐life. Further, we want to know whether LA‐CocHs are effective in blocking the physiological and psychological effects of cocaine. Based on the animal data, we may select the LA‐CocH with the best overall in vivo profile for further development.MethodsStructure and mechanism‐based computational design was followed by laboratory experimental studies. LA‐CocHs were produced in a bioreactor using stable cell lines that we developed previously, and were purified. The purified LA‐CocHs were used to test their actual in vivo activities in animals (rats and monkeys), including pharmacokinetics (PK), pharmacodynamics (PD), hyperactivity, drug discrimination, and drug self‐administration studies.ResultsWe have successfully designed, prepared, and characterized a set of LA‐CocHs, including CocH5‐Fc(6M). According to the animal data (including those from extensive PK/PD, locomotor activity, drug discrimination, and drug self‐administration tests), a single dose of LA‐CocH was able to effectively accelerate cocaine metabolism in animals even after 30 days and, thus, block cocaine‐induced physiological effects and toxicity for a long period of time. In consideration of the general observation that the biological half‐life of a protein drug in humans is generally longer than that in animals, CocH5‐Fc(6M) examined in this study could allow dosing once every month for cocaine addiction treatment in humans.ConclusionThe animal data suggest that CocH5‐Fc(6M) has the desirable potency of a therapeutic enzyme. Hence, CocH5‐Fc(6M) could be developed further to become the desirable enzyme therapy for cocaine addiction treatment.Support or Funding InformationThis work was supported by an NIDA Translational Avant‐Garde Award (grant UH2/UH3 DA041115) and NIDA R01 grants (DA035552, DA032910, DA013930, and DA025100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein kinase C ζ‐dependent upregulation of GDNF by metformin
Authors: Katila Nikita , Choi Dong‐Young , Bhurtel Sunil 
Published: June 2021

Parkinson's disease is the second most common neurodegenerative disorder characterized by prominent loss of the dopaminergic neurons in the substantia nigra (SN). The substantial demise of the dopaminergic neurons in the SN is followed by depletion of dopamine in the striatum manifesting typical motor symptoms including resting tremor, bradykinesia, and rigidity. In spite of effort to develop therapies for the disease, there is still no way to cure or halt the neurodegeneration. Recently, we have demonstrated that metformin rescued the nigrostriatal dopaminergic pathway from MPTP neurotoxicity by inducing neurotrophic factors. In this study, we attempted to address how metformin upregulates neurotrophic factors in the brain which might contribute to the neuroprotection. We observed the increase in the levels of brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) in the SN and striatum of C57BL6 mice via immunohistochemistry and western blotting. These results were recapitulated in vitro study using SH‐SY5Y cells. Upregulation of the neurotrophic factors was accompanied by the activation of AKT/ERK/CREB pathway. Furthermore, we found that inhibition of protein kinase (PK) C ζ attenuated metformin‐induced GDNF elevation, but not BDNF. Tyrosine hydroxylase phosphorylation by metformin was also blocked by PKC ζ inhibition. These results suggest that metformin increase GDNF through induction of PKC ζ activity, which might exert neuroprotection. Although further research is required to identify the metformin's action mechanism, metformin might be a potential neuroprotective therapy for Parkinson's disease treatment.Support or Funding InformationThis work was supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC) grant funded by the Korean Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.