bluuebunny/crossref_metadata_2025_split huggingface dataset:
MED17 is Phosphorylated at S53 by CK2 for Transcriptional Activation of Lipogenic Genes in Response to Insulin
Authors: Viscarra Jose A. , Wang Yuhui , Sul Hei Sook 
Published: June 2021

De novo lipogenesis is under tight regulation by nutritional status and hormonal conditions. Many of the enzymes involved in this process, such as fatty acid synthase (FASN), are regulated at the transcriptional level by insulin signaling cascades. We previously showed that USF1 is a key transcription factor for FASN activation that, by binding to the −65 E‐box, leads to the recruitment of various other lipogenic transcription factors for insulin mediated transcriptional activation. We show here that USF1 directly interacts with the Mediator subunit MED17 at the FASN promoter for recruitment of the Mediator complex, as well as POL II and other general transcription machinery. Additionally, we show that MED17 is phosphorylated at S53 by casein kinase 2 (CK2) in the livers of fed mice or insulin‐stimulated hepatocytes, but not in the livers of fasted mice or untreated hepatocytes, and that this phosphorylation event is required for the FASN promoter activation in response to insulin. To assess whether this newly identified pathway becomes dysregulated in obesity, we next examined phosphorylation of MED17 in the liver of leptin‐deficient and insulin resistant ob/ob mice and, indeed, we detected chronic phosphorylation of S53 even in livers of fasted mice. These results demonstrate that phosphorylation of MED17 at S53 by CK2 is required for the transcriptional activation of lipogenic genes in response to insulin, and furthermore, that dysregulation of this pathway may contribute to the chronic activation of lipogenesis for insulin resistance.Support or Funding InformationRO1DK081098 (NIH)F32DK105671 (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.
A Novel Multi‐domain Phosphatidylinositol Transfer Protein/Oxysterol Binding Protein Senses Specific Phosphoinositide Pools On Toxoplasma Dense Granules
Authors: Grabon Aby , Bankaitis Vytas A. 
Published: June 2021

The Apicomplexan Toxoplasma gondii is a common parasite of humans and other mammals and causes severe disease in immunocompromised individuals. Apicomplexa are obligate intracellular pathogens, and their infection of host cells requires the orchestrated secretion of proteins from several highly specialized secretory organelles, the regulation of which is incompletely understood. Studies in other eukaryotic systems have established that membrane trafficking through distal stages of the secretory pathway is regulated by phosphoinositide (PIP) signaling networks and that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are important components of these signaling circuits. We used PIP‐specific biosensors to map phosphoinositide pools on Toxoplasma membranes, and identified at least two distinct pools of PtdIns(4)P: one on the Golgi/early secretory membranes, and one associated with Apicomplexan‐specific secretory organelles called dense granules (GRA3(+)). Studies in yeast demonstrate that PITPs potentiate the activities of PtdIns kinases (PIKs) to produce privileged PIP pools on secretory membranes, and PtdIns(4)P‐mediated pro‐secretory activities are countered by the antagonistic activities of specific members of the oxysterol binding protein (OSBP) superfamily. We have identified a Toxoplasma multi‐domain protein, the PITP membrane protein (PIMP), that couples a START‐type PITP domain, a pleckstrin homology (PH) domain, and a C‐terminal OSBP domain. PIMP is a bona fide PITP that transfers PtdIns and phosphatidylcholine (PtdCho) between membranes in vitro, and potentiates PIK activity in vivo when expressed in yeast. Of the four known Toxoplasma PITP genes, it is the only one capable of doing so. The PIMP PH domain specifically binds PtdIns(4)P and PtdIns(4,5)P2 in vivo, and recognizes these pools on dense granules in intracellular parasites. Full length PIMP localizes to (GRA3+) dense granules, and this association is mediated by PH domain PIP binding. PIMP is a physical platform for integration of multiple lipid signals on specialized secretory membranes in Toxoplasma and is the first known example of a PITP coupled to an OSBP domain. As dense granules are unique to Apicomplexa and required to support intracellular proliferation of the parasites, dense granule signaling networks would make good therapeutic targets for Apicomplexan infections.Support or Funding InformationThis research was funded by NIH grant R01‐GM112591, RO1‐GM44530 and grant BE‐0017 from the Robert A. Welch Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissection of the Erv41‐Erv46 retrieval pathway suggests a redox‐regulated mechanism
Authors: Keiser Kristofer , Shibuya Aya , Barlowe Charles 
Published: June 2021

The early secretory pathway consists of ER‐ and Golgi‐resident proteins involved in synthesis, folding, quality control, and post translational modifications of secretory proteins. Anterograde flux through this pathway can lead to the mislocalization of ER‐resident proteins as they passively leak to the Golgi. Fortunately there are several retrieval mechanisms to return mislocalized ER‐residents back to the ER. One such retrieval process identified by our lab relies on the Erv41‐Erv46 complex, which recognizes specific non‐HDEL ER‐residents in the Golgi and returns them to the ER in a COPI‐dependent manner. The focus of our work is to determine the mechanism by which the Erv41‐Erv46 retrograde receptor binds to escaped ER‐residents in the Golgi and releases this cargo in the ER.Site‐directed mutagenesis to introduce Erv41 and Erv46 mutations in Saccharomyces cerevisiae revealed key residues involved in retrieval of non‐HDEL ER‐residents. One such series of mutations involves a cysteine rich region of Erv46 where Cys to Ser mutants result in a complete loss of function. This cysteine rich region contains two CXXC motifs typically found in redox proteins. As observed in other redox proteins, the residues between the vicinal cysteines in this region of Erv46 are important suggesting that the redox potential of these cysteines is finely tuned for activity. Furthermore, mutations of a four residue hydrophobic patch within the cysteine rich region as well as a distal basic residue in Erv46 leads to a failure in retrieval of ER‐residents. These residues, but not the cysteines, are conserved in Erv41, however concomitant mutations in Erv41 do not affect retrieval suggesting Erv46 is predominantly involved in binding while Erv41 may play a distinct role.Based on our results, we propose the redox state of Erv46 is highly dynamic resulting in active and inactive disulfide configurations which can expose or mask a cargo binding hydrophobic patch. Defining the Erv41‐Erv46 binding mechanism may allow for a more complete understanding of ER homeostasis as well as providing a potential target to block pathogenic toxins that exploit Erv41‐Erv46 mediated retrieval for efficient delivery to the ER.Support or Funding InformationNIH GM52549This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR‐dependent selective translation rapidly expands lysosome biogenesis, volume and retention capacity during phagocyte activatio
Authors: Botelho Roberto , Hipolito Victoria , Tandoc Kristofferson , Topisirovic Ivan 
Published: June 2021

The molecular mechanisms that govern and adapt the number, size, morphology and activities of organelles to suit the needs of the many cell types and conditions that these cells may encounter are at the frontier of cell biology. Lysosomes are organelles that degrade cargo from various routes including endocytosis, phagocytosis and autophagy. Lysosomes have also emerged as a signalling platform that senses and integrates stress signals such as nutrient deprivation with regulatory kinase hubs like mTOR and AMPK to modulate metabolic activity. For phagocytes and antigen‐presenting cells like macrophages and dendritic cells, lysosomes are a kingpin organelle since they are essential to kill pathogens, and process and ultimately present antigens. During phagocyte activation with lipopolysaccharides, lysosomes undergo a striking reorganization, changing from dozens of globular objects to a tubular network, in a process that requires the phosphatidylinositol‐3‐kinase‐Akt‐mTOR signalling axis. Ultimately, lysosome tubulation is thought to promote pinocytic retention and antigen presentation. Here, we show that lysosome tubulation is accompanied by a rapid boost in lysosome volume and holding capacity of phagocyte activation. We reveal that lysosome expansion depends on biosynthesis of lysosomal proteins but is independent of TFEB and TFE3, transcription factors known to scale up lysosome biogenesis. Instead, we show that lysosome transcripts are selectively targeted for enhanced protein translation in an mTOR‐dependent manner. Indeed, lysosome transcripts were enriched in polyribosome fractions after lipopolysaccharide exposure in an mTOR‐dependent manner, while transcripts encoding non‐lysosomal proteins were unresponsive to LPS and/or mTOR inhibition. Collectively, we identified an example of rapid organelle expansion and remodelling driven by enhanced and selective protein translation during phagocyte activation.Support or Funding InformationCanadian Institutes of Health Research, Canada Research Chair Program, and Ryerson UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the effect of Endoplasmic Reticulum (ER) stress on near‐mitochondrial localized translation
Authors: Maity Shuvadeep , Back Songhee , Rendleman Justin , Vogel Christine 
Published: June 2021

Local synthesis of proteins near their active site is a critical cellular phenomenon. Recent studies have demonstrated that a subpopulation of cytoplasmic ribosomes is localized to organelles such as the endoplasmic reticulum and mitochondria. Due to the presence of different membranous compartments within eukaryotic cells, proper mRNA localization or targeted translation is required to focus the expensive process of translation to where it is needed most. During stress while global translation has been downregulated translation of stress responsive proteins as well other essential proteins remains active. During ER stress, substantial amounts of remodeling happen near ER localized translation populations. Due to its physical and functional relation with mitochondria it is also essential to regulate the translation process of mitochondrial destined proteins during the ER stress condition.In the current study, a digitonin based extraction method has been successfully established to separate the free cytoplasmic ribosome and mitochondria bound ribosome from HEK293 cells and to perform ribosome profiling in order to capture the actively translating polysome populations of free and mitochondria bound translating ribosome populations. This method can successfully separate the two ribosomal populations and it is enriched with known near‐mitochondria localized ribosome. Previous studies identified a population of mRNAs which are translating near mitochondrial surface in yeast and most of them has been found to be conserved in higher organisms. Interestingly, a set of known near mitochondria localized RNA has been found to involve in active translation in our global riboseq data collected from Hela upon ER stress. It indicates a correlation between the near mitochondrial localized translation and ER stress regulation. Specifically, higher expression of different sub units of oxidative phosphorylation complexes has been found to be enriched upon ER stress. This motivates further detailed study to understand organeller cross talk at translation level during stress. Understanding the near mitochondria localized populations in presence and absence of ER stress in higher organisms would be helpful to understand the complex ER‐mitochondrial cross talk events.Support or Funding InformationNIHR01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of excessive mitochondrial fission protects the cardiomyocyte against palmitate‐induced lipotoxicity
Authors: Li Sin‐Jin , Chen Ching‐Yi 
Published: June 2021

Mitochondria play the major role in maintaining the optimal cardiac function. Recently, mitochondrial dynamics have been connected with cardiovascular diseases. Cardiac oxidative stress and imbalanced mitochondrial dynamics (decreased fusion and excessive fission) are involved in lipotoxicity‐induced cardiomyopathy. Our aim was to investigate the role of mitochondrial dynamic in H9c2 cells under lipotoxicity. Treatment of H9c2 cells (cardiomyoblast) with 200 μM palmitate resulted in decreased cell viability and decreased ATP production. Palmitate treatment for 3 h elevated protein expression of the dynamin‐related protein‐1 (DRP1, a mitochondrial fission protein) and PTEN‐induced putative kinase 1 (PINK1, marker of mitophagy), but 24 h incubation with palmitate reduced expression of mitochondrial fusion‐ (mitofusin‐2, Mfn2), fission‐ (DRP1 and fission 1 protein, Fis1) and mitophagy‐ related proteins. To correct the mitochondrial imbalance on the initiation of lipotoxicity, we inhibited Drp1 to evaluate whether this action restores mitochondrial function. Knockdown of DRP1 resulted in decreased apoptosis in cells after palmitate treatment for 24 h. To further determine the functional effects of knockdown of DRP1 on palmitate stress in a short‐term or long‐term period (plamitate treatment for 1 h or 24 h), we measured cellular respiration using Seahorse analyzer. Treatment of H9c2 with plamitate for 1 h reduced the oxygen consumption rate and extracellular acidification rate during basal respiration, whereas inhibition of DRP1 maintained the mitochondrial functions. This result indicated that the energetic function was shifted to quiescence in palmitate‐treated cells whereas knockdown of DRP1 cell did not have this effect. Palmitate treatment resulted in a remarkable reduction ATP production while knockdown of DRP1 restored their reductions. Non‐mitochondrial oxygen consumption (e.g. ROS generation) was upregulated with palmitate treatment, but DPR1 suppression decreased ROS generation. However, there was lower basal respiration, ATP production and FCCP‐stimulated maximal respiration in both groups following 24 h‐palmitate treatment which indicated that DRP1 knockdown could not restore mitochondrial oxidative function. In conclusion, palmitate disrupted mitochondrial dynamic and induced cell death, whereas inhibition of mitochondrial fission (DRP1) at the onset of lipotoxicity maintained the mitochondrial function and cell survival; however, it did not restore mitochondrial function after prolonged palmitate stress, demonstrating its limitations.Support or Funding InformationMOST‐104‐2313‐B‐002‐038‐MY3This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular and structural recognition of Listeria cell‐wall glycopolymers by bacteriophage‐encoded SH3b domains
Authors: Shen Yang , Kalograiaki Ioanna , Prunotto Alessio , Dunne Matthew , Sumrall Eric , Cañada Francisco Javier , Loessner Martin 
Published: June 2021

Targeting carbohydrate recognition plays a key role in multiple biological processes across all kingdoms. SH3b (or bacterial Src homology 3) domain is known as a conserved carbohydrate‐binding module that is responsible for carbohydrate recognition. However, the molecular and structural mechanisms underlying SH3b‐carborhydrate interaction remain elusive. Previously, we reported that three phage‐encoded SH3b domains (termed CBD500, CBDP35 and CBD025) exhibit distinct binding patterns that correlate with structural variations in Listeria wall teichoic acids (WTAs, or glycosylated polyribitol phosphate). Here, we use CBD500 to investigate how it recognizes specific carbohydrate ligand displayed on the bacterial surface. We unveil the contribution of glycosylation and O‐acetylation of WTA polymers to the binding by surface plasmon resonance analysis. Saturation Transfer Difference (STD)‐NMR further substantiates that disparity in GlcNAc to ribitol connectivity, O‐acteyl group, and glycosylated GlcNAc determine the binding specificity. Crystallography, epitope mapping and docking studies identify key residues that form a binding groove (including Phe175, Trp 198, Asn248, and Ser249) mediating CBD500‐WTA interactions, which are validated by site‐directed mutagenesis. This is the first report providing structural insights into the interactions between SH3b and native WTA polymer, thus guiding further studies on structure‐function relationship of SH3b‐carbohydrate complex, as well as support the potential use of endolysins as novel antimicrobials.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dolichol Phosphate Mannose Synthase: A Glycosyltransferase with Unity in Molecular Diversities
Authors: Baksi Krishna , Zhang Zhenbo , Serrano Negron Jesus E. , Banerjee Dipak K. 
Published: June 2021

N‐glycans provide structural and functional stability to asparagine‐linked (N‐linked) glycoprotein, and add flexibility. Glycan biosynthesis is elaborative, multi‐compartmental and involves many glycosyltransferases. Failure to assemble N‐glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N‐glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol‐linked tetra‐decasaccharide (Glc3Man9GlcNAc2‐PP‐Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O‐ and C‐mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP‐dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C‐terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C‐terminus, DPMS has been classified as Type I and Type II enzyme. DPMS belonging to Type II sub‐class has been considered to be a multimeric protein requiring the DPM2 and DPM3 to anchor down the DPM1 to the ER membrane. Our results indicate that the recombinant DPMS from bovine adrenal medullary capillary endothelial cells can be expressed successfully in E. coli as an active enzyme. Furthermore, DPMS is regulated by extracellular signaling and microenvironment. So, we conclude that it is the hydrophobic domain that anchor down DPMS to the ER membrane.Support or Funding InformationSupported in part by the grant NIH/NIMHD G12MD007583 (KB)This 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 FIJI workflow demonstrates dynamic changes in postnatal respiratory nuclei innervation by Nkx2.2‐ and Olig3‐derived neurons.
Authors: Liu Jillian , Fair Summer , Kaya Behiye , Zuniga Jessica , Mostafa Hasnaa , Alves Michele , Czeisler Catherine , Otero Jose 
Published: June 2021

The autonomic nervous system undergoes rapid and significant change in the postnatal period of mammalian development. How the contributions of specific neural lineages to autonomic circuits change and develop during this phase remains to be determined. In this study, we utilized transgenic mouse modeling and immunofluorescent imaging to determine how Nkx2.2‐ and Olig3‐derived neural lineages develop postnatally and, more specifically, how their innervations into three hindbrain respiratory nuclei are altered during this epoch. We generated a novel FIJI workflow, which allowed the systematic and rapid analysis of Nkx2.2‐ and Olig3‐ derived synapses in regions of interest in each of the three hindbrain respiratory nuclei. This workflow provided more precise quantifications in only 1/25 of the time when compared to human quantifications. We demonstrated that Nkx2.2‐derived innervations into the PreBötzinger Complex significantly decreased in the first three weeks of postnatal life while Olig3‐derived afferents showed no such change during the same period. These data demonstrate utility in the application of this novel FIJI workflow to the investigation of neural circuit contributions to the postnatal development of the autonomic nervous system. Further, they indicate that the role of Nkx2.2‐derived neurons in autonomic function and respiration changes during the first three weeks of life.Support or Funding InformationThis work was sponsored by NIH/NHLBI grants R01HL132355, R01HL132355S1, and R01HL132355S2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.