Biochemical Fractionation Research Articles

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.

Environmental controls on the carbon and water (H and O) isotopes in peatland Sphagnum mosses

We conducted a modern process study on Sphagnum magellanicum from southern Patagonian peat bogs to improve our understanding of environmental controls on the carbon and water (hydrogen and oxygen) isotope compositions of peat mosses. We found that moisture availability gradients in peat bogs, measured by Sphagnum water content, could explain the intra-site variability in both Sphagnum cellulose δ13C and δ18O. Also, there was a site-specific significant negative correlation between cellulose δ13C and δ18O across microtopographical gradient. This new finding suggests that Sphagnum moisture availability influences cellulose δ13C via water film effect on discrimination against 13CO2 and, similarly, can imprint on cellulose δ18O via evaporative enrichment of 18O in metabolic leaf water. Sphagnum leaf wax n-alkane δ13C also responded to the water film effect as cellulose did, but n-alkane δ2H appears less sensitive than cellulose δ18O to the moisture availability gradient likely because hydrogen isotopes have a more complex biochemical fractionation pathway and a smaller kinetic fractionation during leaf water evaporation. Furthermore, we used long strands of Sphagnum to explore if isotopic signals in moss strand increments were sensitive to recent growing season conditions. We demonstrated that rapidly growing Sphagnum strand increments could document the isotopic composition of precipitation and moisture conditions at sub-annual scale. Altogether, these findings highlight the sensitivity of stable isotopes in Sphagnum to environmental conditions. On the basis of these results, we propose that paired measurements of carbon and water isotopes in Sphagnum cellulose or leaf wax biomarker provide an improved approach in peat-based paleoclimate reconstructions.

‐The Function of CASK in Transcriptional Regulation in Neurons

CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.Support or Funding InformationThis project has been supported by CABRI Undergraduate Research Fellowship

Screening of microalgae liquid extracts for their bio stimulant properties on plant growth, nutrient uptake and metabolite profile of Solanum lycopersicum L.

The present study investigates the biostimulant effects of 18 Crude Bio-Extracts (CBEs) obtained from Microalgae and Cyanobacteria on tomato plant growth, chlorophyll content, nutrient uptake and metabolite profile. Significant root and shoot length improvement (112.65%, 53.70%); was recorded at treatment with Aphanothece sp and C. ellipsoidea CBEs respectively. Meanwhile, the highest root and shoot dry weight (DW) (34.81%, 58.69%) were obtained at treatment with Aphanothece sp. The latter also displayed the maximum uptake of Nitrogen, phosphorus and potassium, which increased by 185.17%, 119.36% and 78.04% respectively compared with non-treated plants. Principal Component Analysis (PCA) confirmed that Phosphorus and Potassium levels in roots were closely related to enhanced Root length, whereas Nitrogen and chlorophyll b were closely related to Shoot and root DW. Additionally, Gas Chromatography-mass spectrometry (GC-MS) indicated that treatment with CBEs, induced the production of a vast array of metabolites. Treated plants recorded higher accumulation of palmitic and stearic acids, which could indicate a stimulation in de novo Lipid synthesis. CBEs also triggered the accumulation of pyridine-3-carboxamide (an amide active form of vitamin B3) and Linolenic acid; one of the key precursors in the biosynthetic pathway leading to plant jasmonates. Our results are a first step towards understanding the effects of microalgal extracts on plant physiology and biochemical pathways. Further investigations on biochemical fractionation of microalgal extracts and agronomic tests of their purified bioactive compounds could be a useful principal novelty for in-depth study of CBE action mechanisms. Other useful tools include; Comparative hormone profiling of treated and non-treated plants accompanied with combined High-Throughput Plant Phenotyping, transcriptomics and metabolomics analysis.

Chromatin-enriched RNAs mark active and repressive cis-regulation: An analysis of nuclear RNA-seq.

Long noncoding RNAs (lncRNAs) localize in the cell nucleus and influence gene expression through a variety of molecular mechanisms. Chromatin-enriched RNAs (cheRNAs) are a unique class of lncRNAs that are tightly bound to chromatin and putatively function to locally cis-activate gene transcription. CheRNAs can be identified by biochemical fractionation of nuclear RNA followed by RNA sequencing, but until now, a rigorous analytic pipeline for nuclear RNA-seq has been lacking. In this study, we survey four computational strategies for nuclear RNA-seq data analysis and develop a new pipeline, Tuxedo-ch, which outperforms other approaches. Tuxedo-ch assembles a more complete transcriptome and identifies cheRNA with higher accuracy than other approaches. We used Tuxedo-ch to analyze benchmark datasets of K562 cells and further characterize the genomic features of intergenic cheRNA (icheRNA) and their similarity to enhancer RNAs (eRNAs). We quantify the transcriptional correlation of icheRNA and adjacent genes and show that icheRNA is more positively associated with neighboring gene expression than eRNA or cap analysis of gene expression (CAGE) signals. We also explore two novel genomic associations of cheRNA, which indicate that cheRNAs may function to promote or repress gene expression in a context-dependent manner. IcheRNA loci with significant levels of H3K9me3 modifications are associated with active enhancers, consistent with the hypothesis that enhancers are derived from ancient mobile elements. In contrast, antisense cheRNA (as-cheRNA) may play a role in local gene repression, possibly through local RNA:DNA:DNA triple-helix formation.

Characterization of METTL16 as a cytoplasmic RNA binding protein

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16's localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of METTL16 through long-term knockdown.

Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry

Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several other methods used for drug-target identification, such as biochemical fractionation or radioactive ligand binding assay, affinity purification is widely used for its straight-forward and easy approach. To identify the target protein of an inhibitor of cancer cell-accelerated fibroblast migration, we prepared the inhibitor-conjugated beads by photo-crosslinking. Proteins were pulled down from cell lysates by the compound beads and separated by SDS-PAGE, and a specifically pulled down protein was cut out, trypsin-digested, analyzed using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) and identified by peptide mass fingerprinting (PMF) method. Since the photo-crosslinking enables the immobilization of ligands on an affinity matrix in a functional group-independent manner, we do not have to determine the functional group of the compound to conjugate the matrix. In addition, as compared to other MS techniques such as electrospray ionization, MALDI offers a less complex sample preparation procedure and higher sensitivity, and thus is better suited for the rapid identification of proteins isolated by gel electrophoresis.

N-glycosylation of Rim21 at an Unconventional Site Fine-tunes Its Behavior in the Plasma Membrane.

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

A GPR174-CCL21 module imparts sexual dimorphism to humoral immunity.

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174-an X-chromosome-encoded G-protein-coupled receptor-suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume.

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size effector. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. We propose that reductions in cell volume and the amounts of limiting components, such as Npm2, contribute to developmental nuclear size scaling.

Changes in the microsomal proteome of tomato fruit during ripening

The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.

Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism

ObjectiveThe dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways. MethodsWe designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS). ResultsWe show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses. ConclusionsOur data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.

The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress

ABSTRACT The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. Abbreviation AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4′,6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.

Expression Analyses of POGZ, A Responsible Gene for Neurodevelopmental Disorders, during Mouse Brain Development

POGZ is a heterochromatin protein 1 α-binding protein and regulates gene expression. On the other hand, accumulating pieces of evidence indicate that the POGZ gene abnormalities are involved in various neurodevelopmental disorders. In this study, we prepared a specific antibody against POGZ, anti-POGZ, and carried out biochemical and morphological characterization with mouse brain tissues. Western blotting analyses revealed that POGZ is expressed strongly at embryonic day 13 and then gradually decreased throughout the brain development process. In immunohistochemical analyses, POGZ was found to be enriched in cerebrocortical and hippocampal neurons in the early developmental stage. The nuclear expression was also detected in Purkinje cells in cerebellum at postnatal day (P)7 and P15 but disappeared at P30. In primary cultured hippocampal neurons, while POGZ was distributed mainly in the nucleus, it was also visualized in axon and dendrites with partial localization at synapses in consistency with the results obtained in biochemical fractionation analyses. The obtained results suggest that POGZ takes part in the regulation of synaptic function as well as gene expression during brain development.

Arginine vasopressin attenuates dysfunction of hippocampal theta and gamma oscillations in chronic cerebral hypoperfusion via V1a receptor

Chronic cerebral hypoperfusion (CCH) is associated with cognitive decline in aging, Alzheimer's disease and vascular dementia. Neural oscillations and their interactions support brain communication and involve in cognitive function. Although arginine vasopressin (AVP) has been linked to spatial learning and memory, the effects of AVP on CCH in terms of the hippocampal neural network is unknown. Here we investigated the dynamics of neural oscillations in the hippocampus in a rat model of permanent bilateral carotid arteries occlusion (two-vessel occlusion, 2VO) under urethane-anesthesia. Hypertonic saline (5.3%) was injected intraperitoneally to induce the endogenous AVP, and SR49059 was used as V1a receptor (an AVP receptor) antagonist. The results showed that AVP partly changed CA3 Schaffer collateral (CA3-SC) power distribution in the rat model of 2VO via V1a receptor, increased theta synchrony between CA3-SC and CA1 areas, enhanced CA3-SC theta-middle gamma phase-phase coupling, and improved spatial learning and memory performance. Biochemical fractionation further confirmed the recovery effect on N-methyl-D-aspartate receptor subunit 2B (NR2B) and postsynaptic density protein 95 (PSD-95) surface expressions after hypertonic saline injection, suggesting a possible molecular mechanism in the hippocampus. The findings shed light on a functional role of endogenous AVP from a neural network perspective that AVP improves theta synchronization and accurate coordination of theta-gamma coupling probably through upregulating NR2B and PSD-95 expressions, and further promotes neural communication in the hippocampus to some extent. As a result, the impairment of spatial learning and memory induced by CCH is significantly alleviated.

Abstract 505: Adenovirus Targets Connexin43 Gap Junction Expression and Function During Infection

Introduction: Myocarditis leads to pathologic remodeling of the myocardium and arrhythmia. A leading etiological agent of viral myocarditis is human Adenovirus type 5 (Ad5). Individuals affected are at a higher risk of arrhythmogenesis which can lead to sudden cardiac death. Gap junctions, comprised of connexin proteins, provide direct intercellular communication between cardiomyocytes to facilitate electrical coupling. Connexin43 (Cx43) is the predominant connexin expressed in the working myocardium and is tightly regulated by post-translational modifications. Gap junctions are also known to propagate the innate antiviral immune response but the impact of Ad5 infection on Cx43 expression and function is essentially unexplored. Hypothesis: During Ad5 infection Cx43 gap junctions are directly targeted to limit intercellular communication thus precipitating an arrhythmogenic substrate. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), keratinocytes (HaCaT), and lentivirus-transduced HaCaTs stably overexpressing Cx43 were infected with Ad5. Biochemical fractionation and pulse chase assays were implemented to determine gap junction and Cx43 stability. Cx43 protein levels and phosphorylation status were determined by western blotting and immunoprecipitation experiments. Confocal immunofluorescence microscopy was employed determine localization and functional of Cx43 during Ad5 infection. Results: Cx43 expression is targeted early in infection with remaining protein localized at the cell-cell boarder during infection. Phosphorylation of Cx43-Ser368 was detected during early Ad5 infection implicating reduced gap junction conductance. In contrast phosphorylation of Cx43-Ser262 is reduced during Ad5 infection with a concomitant increase in protein half-life despite losses in functional gap junction intercellular communication. Conclusion: Cx43 gap junction structures are stabilized during Ad5 infection but directly targeted by specific phosphorylation events to effect channel closure. Insight provided into mechanisms by which Ad5 manipulates Cx43 regulation will provide novel therapeutic avenues to target gap junction function in a broad spectrum of heart disease states.

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.

Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein–protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.

Atypical chromatin structure of immune-related genes expressed in chicken erythrocytes.

The major biological role of red blood cells is to carry oxygen to the tissues in the body. However, another role of the erythroid cell is to participate in the immune response. Mature erythrocytes from chickens express Toll-like receptors and several cytokines in response to stimulation of the immune system. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from polychromatic erythrocytes from chickens. In conjunction with next-generation DNA, RNA sequencing, chromatin immunoprecipitation-DNA sequencing, and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing, we identified the active chromosomal compartments and determined their structural signatures in relation to expression levels. Here, we present the detailed chromatin characteristics of erythroid genes participating in the innate immune response. Our studies revealed an atypical chromatin structure for several genes coding for Toll-like receptors, interleukins, and interferon regulatory factors. The body of these genes had nucleosome-free regions intermingled with nucleosomes modified with H3K4me3 and H3K27ac, suggesting a dynamic unstable chromatin structure. We further show that human genes involved in cell identity have gene bodies with the same chromatin-instability features as the chicken polychromatic erythrocyte genes participating in the innate immune response.

Abstract 1201: IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer

Abstract IL-4 has a number of well-established functions in immune cells including proliferation, and survival of lymphocytes, polarization of macrophages to the pro-tumor M2 tumor associated macrophage phenotype. Another role of IL-4 signaling is induction of metabolic changes in immune cells including increased glycolysis in B-lymphocytes, and increased expression of arginase 1 with a switch to fatty acid oxidation in macrophages. However, the effects of IL-4 signaling in tumor cells that express IL-4 receptor is not well understood. Previous work from our lab has shown that loss of IL-4 receptor (IL-4R) significantly abrogates metastasis in experimental metastasis models in mice. Thus, we wondered whether changes in metabolism downstream of IL-4 receptor activation may contribute to metastatic phenotypes. Since altered metabolic pathways have been shown to regulate the epigenetic state of cancer cells, we are pursuing histone modifications as a mechanism for the connection between altered metabolism and increased metastatic capability in IL-4R expressing breast cancer cells. MDA-MB-231 and BT549 triple negative breast cancer cells were treated with IL-4 for 2,4,6, and 8 days. Samples of media were assayed for glutamine and glucose consumption, as well as lactate and glutamate secretion. IL-4 decreased consumption of glucose and glutamine, yet concomitantly increased the release of lactate and glutamate. These data suggest that IL-4 alters the utilization of glucose and glutamine in cancer cells. To study potential links to epigenetic regulation cells were treated for 18, or 48 hours with IL-4 before biochemical fractionation was performed in order to obtain nuclear and cytosolic lysates. The nuclear fractions were assessed for histone acetyltransferase (HAT) activity using commercially available assays, and showed that IL-4 increased the activity of histone acetyltransferases. We also performed pull down of acetylated lysine residue from the nuclear fractions of IL-4 treated cells. The cells were then assessed for abundance of acetylated histone 3. In addition, we used western blotting to measure the abundance of specific histone modifications such as histone 3 lysine 9 acetylation and histone 3 lysine 27 acetylation. Overall, histone acetyltransferase activity and levels of histone acetylation were increased by IL-4. Additionally, we found that IL4 increases the acetylation of histone 3 lysine 9. In order to determine the effects of IL-4 signaling on metabolism in vivo we have initiated PET imaging studies in humanized mice that underwent tail vein injections of BT549 cell lines. These mice were treated with MDNA413, an antagonist of IL-4 signaling, and are being assessed for uptake of glucose and glutamine. Together these suggest that IL-4 alters the metabolism and epigenetic state of cancer cells and may be a viable target for therapy in metastatic breast cancer. Citation Format: Demond Williams, Adam Miranda, Chilesi Uririri, Barbara Fingleton. IL4 receptor mediated metabolic changes contribute to histone acetylation leading to metastatic progression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1201.