Publications by authors named "Kyung-Mee Moon"

33 Publications

Proteomic analysis of metabolic pathways supports chloroplast-mitochondria cross-talk in a Cu-limited diatom.

Plant Direct 2022 Jan 20;6(1):e376. Epub 2022 Jan 20.

Department of Earth Ocean and Atmospheric Science University of British Columbia Vancouver British Columbia Canada.

Diatoms are one of the most successful phytoplankton groups in our oceans, being responsible for over 20% of the Earth's photosynthetic productivity. Their chimeric genomes have genes derived from red algae, green algae, bacteria, and heterotrophs, resulting in multiple isoenzymes targeted to different cellular compartments with the potential for differential regulation under nutrient limitation. The resulting interactions between metabolic pathways are not yet fully understood. We previously showed how acclimation to Cu limitation enhanced susceptibility to overreduction of the photosynthetic electron transport chain and its reorganization to favor photoprotection over light harvesting in the oceanic diatom (Hippmann et al., 2017, 10.1371/journal.pone.0181753). In order to gain a better understanding of the overall metabolic changes that help alleviate the stress of Cu limitation, we have further analyzed the comprehensive proteomic datasets generated in that study to identify differentially expressed proteins involved in carbon, nitrogen, and oxidative stress-related metabolic pathways. Metabolic pathway analysis showed integrated responses to Cu limitation. The upregulation of ferredoxin (Fdx) was correlated with upregulation of plastidial Fdx-dependent isoenzymes involved in nitrogen assimilation as well as enzymes involved in glutathione synthesis, thus suggesting an integration of nitrogen uptake and metabolism with photosynthesis and oxidative stress resistance. The differential expression of glycolytic isoenzymes located in the chloroplast and mitochondria may enable them to channel both excess electrons and/or ATP between these compartments. An additional support for chloroplast-mitochondrial cross-talk is the increased expression of chloroplast and mitochondrial proteins involved in the proposed malate shunt under Cu limitation.
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http://dx.doi.org/10.1002/pld3.376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8777261PMC
January 2022

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2.

Sci Rep 2021 12 8;11(1):23630. Epub 2021 Dec 8.

Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein-protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.
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http://dx.doi.org/10.1038/s41598-021-02795-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8655068PMC
December 2021

The parasite Schistocephalus solidus secretes proteins with putative host manipulation functions.

Parasit Vectors 2021 Aug 28;14(1):436. Epub 2021 Aug 28.

Département de Biologie, Université Laval, Quebec, QC, Canada.

Background: Manipulative parasites are thought to liberate molecules in their external environment, acting as manipulation factors with biological functions implicated in their host's physiological and behavioural alterations. These manipulation factors are part of a complex mixture called the secretome. While the secretomes of various parasites have been described, there is very little data for a putative manipulative parasite. It is necessary to study the molecular interaction between a manipulative parasite and its host to better understand how such alterations evolve.

Methods: Here, we used proteomics to characterize the secretome of a model cestode with a complex life cycle based on trophic transmission. We studied Schistocephalus solidus during the life stage in which behavioural changes take place in its obligatory intermediate fish host, the threespine stickleback (Gasterosteus aculeatus). We produced a novel genome sequence and assembly of S. solidus to improve protein coding gene prediction and annotation for this parasite. We then described the whole worm's proteome and its secretome during fish host infection using LC-MS/MS.

Results: A total of 2290 proteins were detected in the proteome of S. solidus, and 30 additional proteins were detected specifically in the secretome. We found that the secretome contains proteases, proteins with neural and immune functions, as well as proteins involved in cell communication. We detected receptor-type tyrosine-protein phosphatases, which were reported in other parasitic systems to be manipulation factors. We also detected 12 S. solidus-specific proteins in the secretome that may play important roles in host-parasite interactions.

Conclusions: Our results suggest that S. solidus liberates molecules with putative host manipulation functions in the host and that many of them are species-specific.
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http://dx.doi.org/10.1186/s13071-021-04933-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8400842PMC
August 2021

The application of forensic proteomics to identify an unknown snake venom in a deceased toddler.

Forensic Sci Int 2021 May 4;323:110820. Epub 2021 May 4.

Michael Smith Laboratories and Department of Biochemistry & Molecular Biology, University of British Columbia, 2185 E Mall, Vancouver, BC V6T 1Z4, Canada. Electronic address:

Proteomics is the global analysis of proteins in a sample, and its methodologies are commonly applied in life science research. Despite its wide applicability however, proteomics is rarely used as a tool in criminal investigations. Here we present a case where the technique provided key evidence in a case that involved the death of a two-year old girl. The defendant was known to keep exotic snakes, including several venomous species, which led the coroner to probe whether there could be snake venom in the blood of the deceased. One major challenge of the investigation was the overwhelming presence of several blood proteins, such as apolipoprotein and complement proteins, which hinders the detection of less abundant analytes. In a counter-acting strategy, a combination of immunodepletion and fractionation methods was used; the sample was then submitted to tandem mass spectrometry for peptide identification. Using this strategy, 15,000 peptides could be sequenced. However, the subsequent challenge was to differentiate between human and snake proteins, given the genetic similarities that are shared by the two vertebrate species. After a thorough bioinformatics search and manual inspection, we found that<1% of the sequenced peptides could be matched unequivocally to snake proteins, including a well-known venom component, phospholipase A2. This evidence, in part, led to a court-issued search warrant of the defendant's home, followed by his arrest and an eventual guilty plea with formal sentencing to 18 months in prison. The work outlined here is an example of how proteomics technology can help to expand the toolkit for molecular forensics.
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http://dx.doi.org/10.1016/j.forsciint.2021.110820DOI Listing
May 2021

Urinary epidermal growth factor is a novel biomarker for early diagnosis of antibody mediated kidney allograft rejection: A urinary proteomics analysis.

J Proteomics 2021 05 27;240:104208. Epub 2021 Mar 27.

Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Electronic address:

Although antibody mediated rejection (AMR) accounts for 20-30% of all acute renal allograft rejections, introducing biomarkers for a timely detection of allograft rejection has been remained challenging. This study investigated novel diagnostic biomarkers of AMR by examining of urine proteome in renal transplant patients. Thirty-six patients with kidney transplantation including 22 AMR patients and 14 patients with stable renal function (control group) were enrolled in this study. Urinary samples were collected and Label free quantification (LFQ) proteomics technique was applied on urine samples and data was subjected to Random Forest (RF) algorithm to predict main candidate proteins contributing in AMR. Finally, applicability of candidate diagnostic biomarkers was evaluated in new sets of AMR subjects, stable patients and healthy volunteers. A total of 1020 proteins were detected in urine proteome. RF algorithm predicted 20 differentially expressed proteins with the highest sensitivity and specificity and combination of EGF, COL6A, and NID-1 was identified as possible panel for early diagnosis of AMR. Applicability of EGF as diagnostic biomarker was validated in urine samples of independent set of AMR subjects. This is the first urinary proteomics study in AMR patients demonstrating that urinary EGF might be used as early diagnostic biomarker for AMR. SIGNIFICANCE: Renal antibody mediated rejection (AMR) accounts for 20-30% of all acute rejections of allografted kidneys. Although several possible biomarkers have been proposed to predict AMR, ineffectiveness of current urinary biomarkers in early diagnosing of AMR patients and in distinguishing AMR subjects from patients with stable kidney function casts doubts on their applicability in clinic. Here for the first time and based on the analysis of urinary proteome we showed that uEGF and uEGF/Cr might be candidate biomarkers to predict AMR with high diagnostic power.
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http://dx.doi.org/10.1016/j.jprot.2021.104208DOI Listing
May 2021

Frequent Assembly of Chimeric Complexes in the Protein Interaction Network of an Interspecies Yeast Hybrid.

Mol Biol Evol 2021 04;38(4):1384-1401

Département de Biochimie, Microbiologie et Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec, QC, Canada.

Hybrids between species often show extreme phenotypes, including some that take place at the molecular level. In this study, we investigated the phenotypes of an interspecies diploid hybrid in terms of protein-protein interactions inferred from protein correlation profiling. We used two yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum, which are interfertile, but yet have proteins diverged enough to be differentiated using mass spectrometry. Most of the protein-protein interactions are similar between hybrid and parents, and are consistent with the assembly of chimeric complexes, which we validated using an orthogonal approach for the prefoldin complex. We also identified instances of altered protein-protein interactions in the hybrid, for instance, in complexes related to proteostasis and in mitochondrial protein complexes. Overall, this study uncovers the likely frequent occurrence of chimeric protein complexes with few exceptions, which may result from incompatibilities or imbalances between the parental proteomes.
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http://dx.doi.org/10.1093/molbev/msaa298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042767PMC
April 2021

Functional Analysis of BipA in E. coli Reveals the Natural Plasticity of 50S Subunit Assembly.

J Mol Biol 2020 09 22;432(19):5259-5272. Epub 2020 Jul 22.

Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA; Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

BipA is a conserved translational GTPase of bacteria recently implicated in ribosome biogenesis. Here we show that Escherichia coli ΔbipA cells grown at suboptimal temperature accumulate immature large subunit particles missing several proteins. These include L17 and L17-dependent binders, suggesting that structural block 3 of the subunit folds late in the assembly process. Parallel analysis of the control strain revealed accumulation of nearly identical intermediates, albeit at lower levels, suggesting qualitatively similar routes of assembly. This came as a surprise, because earlier analogous studies of wild-type E. coli showed early binding of L17. Further investigation showed that the main path of 50S assembly differs depending on conditions of growth. Either supplementation of the media with lysine and arginine or suboptimal temperature appears to delay block 3 folding, demonstrating the flexible nature of the assembly process. We also show that the variant BipA-H78A fails to rescue phenotypes of the ΔbipA strain, indicating a critical role for GTP hydrolysis in BipA function. In fact, BipA-H78A confers a dominant negative phenotype in wild-type cells. Controlled production of BipA-H78A causes accumulation of 70S monosomes at the expense of polysomes, suggesting that the growth defect stems from a shutdown of translation.
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http://dx.doi.org/10.1016/j.jmb.2020.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502552PMC
September 2020

Identification of novel blood biomarkers of treatment response in cystic fibrosis pulmonary exacerbations by label-free quantitative proteomics.

Sci Rep 2019 11 20;9(1):17126. Epub 2019 Nov 20.

Centre for Heart Lung Innovation, University of British Columbia, Vancouver, BC, Canada.

Pulmonary exacerbations (PEx) are clinically impactful events for individuals with CF. Unfortunately, many CF individuals with PEx fail to regain their baseline lung function despite treatment. The objective of this study was to use unbiased proteomic technology to identify novel blood protein biomarkers that change following intravenous (IV) antibiotic treatment and to explore if changes correlate with clinical response by the end of treatment. Blood samples from 25 PEx events derived from 22 unique CF adults were collected within 24 hours of hospital admission, day 5, day 10, and IV antibiotic completion. Three-hundred and forty-six blood proteins were evaluated with label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative proteomics and immunoassays. Forty-seven plasma proteins changed significantly following 5 days of IV antibiotic treatment (q-value ≤ 0.10). Early change in IGF2R from hospital admission to day 5 correlated with overall change in symptom score (CFRSD-CRISS) by the end of treatment (r = -0.48, p-value = 0.04). Several plasma proteins identified and quantified by label-free LC-MS/MS changed early following treatment with IV antibiotics and many of these proteins are involved in complement activation and inflammatory/immune-related pathways. Early change in IGF2R correlated with symptom response following IV antibiotic treatment and requires further validation as a predictive biomarker of symptom response.
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http://dx.doi.org/10.1038/s41598-019-53759-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868239PMC
November 2019

Integrative Genomics Reveals the Genetics and Evolution of the Honey Bee's Social Immune System.

Genome Biol Evol 2019 03;11(3):937-948

Department of Biology, York University, Toronto, Ontario, Canada.

Social organisms combat pathogens through individual innate immune responses or through social immunity-behaviors among individuals that limit pathogen transmission within groups. Although we have a relatively detailed understanding of the genetics and evolution of the innate immune system of animals, we know little about social immunity. Addressing this knowledge gap is crucial for understanding how life-history traits influence immunity, and identifying if trade-offs exist between innate and social immunity. Hygienic behavior in the Western honey bee, Apis mellifera, provides an excellent model for investigating the genetics and evolution of social immunity in animals. This heritable, colony-level behavior is performed by nurse bees when they detect and remove infected or dead brood from the colony. We sequenced 125 haploid genomes from two artificially selected highly hygienic populations and a baseline unselected population. Genomic contrasts allowed us to identify a minimum of 73 genes tentatively associated with hygienic behavior. Many genes were within previously discovered QTLs associated with hygienic behavior and were predictive of hygienic behavior within the unselected population. These genes were often involved in neuronal development and sensory perception in solitary insects. We found that genes associated with hygienic behavior have evidence of positive selection within honey bees (Apis), supporting the hypothesis that social immunity contributes to fitness. Our results indicate that genes influencing developmental neurobiology and behavior in solitary insects may have been co-opted to give rise to a novel and adaptive social immune phenotype in honey bees.
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http://dx.doi.org/10.1093/gbe/evz018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447389PMC
March 2019

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1) ALS mice originate from astrocytes and neurons and carry misfolded SOD1.

J Biol Chem 2019 03 11;294(10):3744-3759. Epub 2019 Jan 11.

From the Centre for Brain Health, Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1B5, Canada,

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1), revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1 mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1 mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1 ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.
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http://dx.doi.org/10.1074/jbc.RA118.004825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416428PMC
March 2019

Transcriptomic and proteomic host response to Aspergillus fumigatus conidia in an air-liquid interface model of human bronchial epithelium.

PLoS One 2018 27;13(12):e0209652. Epub 2018 Dec 27.

Centre for Heart Lung Innovation, University of British Columbia and St. Paul's Hospital, Vancouver, Canada.

Aspergillus fumigatus (A. fumigatus) is a wide-spread fungus that is a potent allergen in hypersensitive individuals but also an opportunistic pathogen in immunocompromised patients. It reproduces asexually by releasing airborne conidiospores (conidia). Upon inhalation, fungal conidia are capable of reaching the airway epithelial cells (AECs) in bronchial and alveolar tissues. Previous studies have predominantly used submerged monolayer cultures for studying this host-pathogen interaction; however, these cultures do not recapitulate the mucocililary differentiation phenotype of the in vivo epithelium in the respiratory tract. Thus, the aim of this study was to use well-differentiated primary human bronchial epithelial cells (HBECs) grown at the air-liquid interface (ALI) to determine their transcriptomic and proteomic responses following interaction with A. fumigatus conidia. We visualized conidial interaction with HBECs using confocal laser scanning microscopy (CLSM), and applied NanoString nCounter and shotgun proteomics to assess gene expression changes in the human cells upon interaction with A. fumigatus conidia. Western blot analysis was used to assess the expression of top three differentially expressed proteins, CALR, SET and NUCB2. CLSM showed that, unlike submerged monolayer cultures, well-differentiated ALI cultures of primary HBECs were estimated to internalize less than 1% of bound conidia. Nevertheless, transcriptomic and proteomic analyses revealed numerous differentially expressed host genes; these were enriched for pathways including apoptosis/autophagy, translation, unfolded protein response and cell cycle (up-regulated); complement and coagulation pathways, iron homeostasis, nonsense mediated decay and rRNA binding (down-regulated). CALR and SET were confirmed to be up-regulated in ALI cultures of primary HBECs upon exposure to A. fumigatus via western blot analysis. Therefore, using transcriptomics and proteomics approaches, ALI models recapitulating the bronchial epithelial barrier in the conductive zone of the respiratory tract can provide novel insights to the molecular response of bronchial epithelial cells upon exposure to A. fumigatus conidia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209652PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307744PMC
May 2019

IRES-dependent ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection.

Nucleic Acids Res 2018 12;46(22):11952-11967

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses 37 nucleotides downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.
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http://dx.doi.org/10.1093/nar/gky1121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294563PMC
December 2018

Integrated systems analysis reveals conserved gene networks underlying response to spinal cord injury.

Elife 2018 10 2;7. Epub 2018 Oct 2.

Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada.

Spinal cord injury (SCI) is a devastating neurological condition for which there are currently no effective treatment options to restore function. A major obstacle to the development of new therapies is our fragmentary understanding of the coordinated pathophysiological processes triggered by damage to the human spinal cord. Here, we describe a systems biology approach to integrate decades of small-scale experiments with unbiased, genome-wide gene expression from the human spinal cord, revealing a gene regulatory network signature of the pathophysiological response to SCI. Our integrative analyses converge on an evolutionarily conserved gene subnetwork enriched for genes associated with the response to SCI by small-scale experiments, and whose expression is upregulated in a severity-dependent manner following injury and downregulated in functional recovery. We validate the severity-dependent upregulation of this subnetwork in rodents in primary transcriptomic and proteomic studies. Our analysis provides systems-level view of the coordinated molecular processes activated in response to SCI.
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http://dx.doi.org/10.7554/eLife.39188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173583PMC
October 2018

Clptm1 Limits Forward Trafficking of GABA Receptors to Scale Inhibitory Synaptic Strength.

Neuron 2018 02 25;97(3):596-610.e8. Epub 2018 Jan 25.

Djavad Mowafaghian Centre for Brain Health and Department of Psychiatry, University of British Columbia, Vancouver, BC V6T 2B5, Canada. Electronic address:

In contrast with numerous studies of glutamate receptor-associated proteins and their involvement in the modulation of excitatory synapses, much less is known about mechanisms controlling postsynaptic GABA receptor (GABAR) numbers. Using tandem affinity purification from tagged GABAR γ2 subunit transgenic mice and proteomic analysis, we isolated several GABAR-associated proteins, including Cleft lip and palate transmembrane protein 1 (Clptm1). Clptm1 interacted with all GABAR subunits tested and promoted GABAR trapping in the endoplasmic reticulum. Overexpression of Clptm1 reduced GABAR-mediated currents in a recombinant system, in cultured hippocampal neurons, and in brain, with no effect on glycine or AMPA receptor-mediated currents. Conversely, knockdown of Clptm1 increased phasic and tonic inhibitory transmission with no effect on excitatory synaptic transmission. Furthermore, altering the expression level of Clptm1 mimicked activity-induced inhibitory synaptic scaling. Thus, in complement to other GABAR-associated proteins that promote receptor surface expression, Clptm1 limits GABAR forward trafficking and regulates inhibitory homeostatic plasticity.
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http://dx.doi.org/10.1016/j.neuron.2017.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810584PMC
February 2018

Contrasting effects of copper limitation on the photosynthetic apparatus in two strains of the open ocean diatom Thalassiosira oceanica.

PLoS One 2017 24;12(8):e0181753. Epub 2017 Aug 24.

Department of Earth, Ocean, and Atmospheric Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

There is an intricate interaction between iron (Fe) and copper (Cu) physiology in diatoms. However, strategies to cope with low Cu are largely unknown. This study unveils the comprehensive restructuring of the photosynthetic apparatus in the diatom Thalassiosira oceanica (CCMP1003) in response to low Cu, at the physiological and proteomic level. The restructuring results in a shift from light harvesting for photochemistry-and ultimately for carbon fixation-to photoprotection, reducing carbon fixation and oxygen evolution. The observed decreases in the physiological parameters Fv/Fm, carbon fixation, and oxygen evolution, concomitant with increases in the antennae absorption cross section (σPSII), non-photochemical quenching (NPQ) and the conversion factor (φe:C/ηPSII) are in agreement with well documented cellular responses to low Fe. However, the underlying proteomic changes due to low Cu are very different from those elicited by low Fe. Low Cu induces a significant four-fold reduction in the Cu-containing photosynthetic electron carrier plastocyanin. The decrease in plastocyanin causes a bottleneck within the photosynthetic electron transport chain (ETC), ultimately leading to substantial stoichiometric changes. Namely, 2-fold reduction in both cytochrome b6f complex (cytb6f) and photosystem II (PSII), no change in the Fe-rich PSI and a 40- and 2-fold increase in proteins potentially involved in detoxification of reactive oxygen species (ferredoxin and ferredoxin:NADP+ reductase, respectively). Furthermore, we identify 48 light harvesting complex (LHC) proteins in the publicly available genome of T. oceanica and provide proteomic evidence for 33 of these. The change in the LHC composition within the antennae in response to low Cu underlines the shift from photochemistry to photoprotection in T. oceanica (CCMP1003). Interestingly, we also reveal very significant intra-specific strain differences. Another strain of T. oceanica (CCMP 1005) requires significantly higher Cu concentrations to sustain both its maximal and minimal growth rate compared to CCMP 1003. Under low Cu, CCMP 1005 decreases its growth rate, cell size, Chla and total protein per cell. We argue that the reduction in protein per cell is the main strategy to decrease its cellular Cu requirement, as none of the other parameters tested are affected. Differences between the two strains, as well as differences between the well documented responses to low Fe and those presented here in response to low Cu are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181753PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570362PMC
October 2017

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

Sci Rep 2017 08 21;7(1):8381. Epub 2017 Aug 21.

Department of Biochemistry & Molecular Biology, and Centre for Sustainable Food Systems, University of British Columbia, Vancouver, BC, Canada.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.
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http://dx.doi.org/10.1038/s41598-017-08464-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566959PMC
August 2017

Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages.

Med Microbiol Immunol 2017 Jun 11;206(3):235-257. Epub 2017 Mar 11.

Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24 h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.
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http://dx.doi.org/10.1007/s00430-017-0500-7DOI Listing
June 2017

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

PLoS One 2017 30;12(1):e0170068. Epub 2017 Jan 30.

Departments of Medicine, University of British Columbia, Vancouver, BC, Canada.

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170068PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5279761PMC
August 2017

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome.

Proc Natl Acad Sci U S A 2017 01 17;114(5):980-985. Epub 2017 Jan 17.

Department of Microbiology, The Ohio State University, Columbus, OH 43210;

The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles-S3, S10, S14, and S21-all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increased levels of precursor 17S rRNA in assembly intermediates. Mutation ΔlepA confers a synthetic growth phenotype in absence of RsgA, another GTPase, well known to act in 30S subunit assembly. Analysis of the ΔrsgA strain reveals accumulation of intermediates that resemble those seen in the absence of LepA. These data suggest that RsgA and LepA play partially redundant roles to ensure efficient 30S assembly.
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http://dx.doi.org/10.1073/pnas.1613665114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293072PMC
January 2017

Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein.

J Biol Chem 2016 May 29;291(21):11300-12. Epub 2016 Mar 29.

From the Departments of Cellular and Physiological Sciences,

Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.
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http://dx.doi.org/10.1074/jbc.M116.714378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900275PMC
May 2016

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis.

PLoS One 2016 16;11(2):e0149177. Epub 2016 Feb 16.

Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Canada.

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149177PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755616PMC
July 2016

Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation.

Cell Metab 2016 Jan 25;23(1):179-93. Epub 2015 Nov 25.

Department of Cellular and Physiological Sciences, Diabetes Research Group, Life Sciences Institute, University of British Columbia, BC V6T1Z3, Canada. Electronic address:

Pancreatic β cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive β cells into more pliable states with reduced cellular insulin levels, increased β cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, β cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner.
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http://dx.doi.org/10.1016/j.cmet.2015.10.016DOI Listing
January 2016

Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

J Neurochem 2016 Jan 17;136(2):276-84. Epub 2015 Nov 17.

Centre for Biological Sciences, University of Southampton, Southampton, UK.

Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.
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http://dx.doi.org/10.1111/jnc.13407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843953PMC
January 2016

Mass Spectrometry-Based Proteomics Identification of Enteropathogenic Escherichia coli Pedestal Constituents.

J Proteome Res 2015 Jun 4;14(6):2520-7. Epub 2015 May 4.

Enteropathogenic Escherichia coli (EPEC) co-opt host signaling pathways and recruit numerous host proteins to motile morphological structures, called pedestals, at sites of bacterial attachment. These pedestals are hallmarks of EPEC-based disease, and the identification and characterization of the functions of pedestal proteins continue to steadily increase. To identify additional constituents in an unbiased manner, we developed a strategy where EPEC pedestals were elongated artificially, severed, and then concentrated prior to their analysis by mass spectrometry (MS)-based proteomics. We identified >90 unique mammalian proteins over multiple experimental trials from our preparations. Seventeen predicted molecules were significantly higher in abundance (p < 0.05) when compared to both the negative controls and sample means. Validation of two identified proteins (cyclophilin A [nonactin-associated] and transgelin [actin-associated]) by immunolocalization was used to confirm our analysis, and both showed enrichment at EPEC pedestals. The EPEC pedestal concentration technique developed here together with the identification of novel pedestal proteins not only provides a resource for the further characterization of molecular components within these structures but also demonstrates that EPEC pedestals can be used as a model system for the identification of novel functions of proteins not normally thought to be at actin-based structures.
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http://dx.doi.org/10.1021/acs.jproteome.5b00074DOI Listing
June 2015

Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

DNA Repair (Amst) 2015 Jun 19;30:68-79. Epub 2015 Feb 19.

Centre de recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada. Electronic address:

An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.
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http://dx.doi.org/10.1016/j.dnarep.2015.02.004DOI Listing
June 2015

A search for protein biomarkers links olfactory signal transduction to social immunity.

BMC Genomics 2015 Feb 8;16:63. Epub 2015 Feb 8.

Department of Biochemistry & Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, BC, V6T 1Z4, Canada.

Background: The Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees.

Results: After confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect.

Conclusions: Our data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens.
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http://dx.doi.org/10.1186/s12864-014-1193-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342888PMC
February 2015

Outer membrane proteins preferentially load MHC class II peptides: implications for a Chlamydia trachomatis T cell vaccine.

Vaccine 2015 Apr 1;33(18):2159-66. Epub 2015 Mar 1.

Vaccine Research Laboratory, University of British Columbia Centre for Disease Control, Vancouver, BC, Canada. Electronic address:

CD4 T cell immune responses such as interferon-γ and tumor necrosis factor-α secretion are necessary for Chlamydia immunity. We used an immunoproteomic approach in which Chlamydia trachomatis and Chlamydia muridarum-derived peptides presented by MHC class II molecules on the surface of infected dendritic cells (DCs) were identified by tandem mass spectrometry using bone marrow derived DCs (BMDCs) from mice of different MHC background. We first compared the C. muridarum immunoproteome in C3H mice to that previously identified in C57BL/6 mice. Fourteen MHC class II binding peptides from 11 Chlamydia proteins were identified from C3H infected BMDCs. Two C. muridarum proteins overlapped between C3H and C57B/6 mice and both were polymorphic membrane proteins (Pmps) which presented distinct class II binding peptides. Next we studied DCs from C57BL/6 mice infected with the human strain, C. trachomatis serovar D. Sixty MHC class II binding peptides derived from 27 C. trachomatis proteins were identified. Nine proteins were orthologous T cell antigens between C. trachomatis and C. muridarum and 2 of the nine were Pmps which generated MHC class II binding epitopes at distinct sequences within the proteins. As determined by antigen specific splenocyte responses outer membrane proteins PmpF, -G and -H and the major outer membrane protein (MOMP) were antigenic in mice previously infected with C. muridarum or C. trachomatis. Furthermore a recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with MOMP formulated with a Th1 polarizing adjuvant significantly accelerated (p<0.001) clearance in the C57BL/6 mice C. trachomatis transcervical infection model. We conclude that Chlamydia outer membrane proteins are important T cell antigens useful in the development of a C. trachomatis subunit vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2015.02.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390527PMC
April 2015

hnRNP K coordinates transcriptional silencing by SETDB1 in embryonic stem cells.

PLoS Genet 2015 Jan 22;11(1):e1004933. Epub 2015 Jan 22.

Life Sciences Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs.
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http://dx.doi.org/10.1371/journal.pgen.1004933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303303PMC
January 2015

A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2.

J Biol Chem 2014 Oct 4;289(42):29350-64. Epub 2014 Sep 4.

From the Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver V6T 2B5, Canada,

Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His6-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His6-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.
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http://dx.doi.org/10.1074/jbc.M114.549279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200284PMC
October 2014
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