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Proteomes, Volume 6, Issue 4 (December 2018)

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Open AccessArticle Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization
Received: 12 October 2018 / Revised: 10 December 2018 / Accepted: 13 December 2018 / Published: 17 December 2018
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Abstract
Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling
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Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessFeature PaperArticle Actinobaculum massiliense Proteome Profiled in Polymicrobial Urethral Catheter Biofilms
Received: 10 October 2018 / Revised: 27 November 2018 / Accepted: 3 December 2018 / Published: 9 December 2018
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Abstract
Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis
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Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as A. massiliense. We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to A. massiliense fitness and persistence in the CBs. Full article
(This article belongs to the Special Issue Proteomic Analysis of Host-Microbial Pathogen Interactions)
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Open AccessFeature PaperReview Cell-Type-Specific Proteomics: A Neuroscience Perspective
Received: 13 November 2018 / Revised: 4 December 2018 / Accepted: 5 December 2018 / Published: 9 December 2018
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Abstract
Cell-type-specific analysis has become a major focus for many investigators in the field of neuroscience, particularly because of the large number of different cell populations found in brain tissue that play roles in a variety of developmental and behavioral disorders. However, isolation of
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Cell-type-specific analysis has become a major focus for many investigators in the field of neuroscience, particularly because of the large number of different cell populations found in brain tissue that play roles in a variety of developmental and behavioral disorders. However, isolation of these specific cell types can be challenging due to their nonuniformity and complex projections to different brain regions. Moreover, many analytical techniques used for protein detection and quantitation remain insensitive to the low amounts of protein extracted from specific cell populations. Despite these challenges, methods to improve proteomic yield and increase resolution continue to develop at a rapid rate. In this review, we highlight the importance of cell-type-specific proteomics in neuroscience and the technical difficulties associated. Furthermore, current progress and technological advancements in cell-type-specific proteomics research are discussed with an emphasis in neuroscience. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessFeature PaperReview From Synapse to Function: A Perspective on the Role of Neuroproteomics in Elucidating Mechanisms of Drug Addiction
Received: 13 November 2018 / Revised: 5 December 2018 / Accepted: 7 December 2018 / Published: 9 December 2018
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Abstract
Drug addiction is a complex disorder driven by dysregulation in molecular signaling across several different brain regions. Limited therapeutic options currently exist for treating drug addiction and related psychiatric disorders in clinical populations, largely due to our incomplete understanding of the molecular pathways
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Drug addiction is a complex disorder driven by dysregulation in molecular signaling across several different brain regions. Limited therapeutic options currently exist for treating drug addiction and related psychiatric disorders in clinical populations, largely due to our incomplete understanding of the molecular pathways that influence addiction pathology. Recent work provides strong evidence that addiction-related behaviors emerge from the convergence of many subtle changes in molecular signaling networks that include neuropeptides (neuropeptidome), protein-protein interactions (interactome) and post-translational modifications such as protein phosphorylation (phosphoproteome). Advancements in mass spectrometry methodology are well positioned to identify these novel molecular underpinnings of addiction and further translate these findings into druggable targets for therapeutic development. In this review, we provide a general perspective of the utility of novel mass spectrometry-based approaches for addressing critical questions in addiction neuroscience, highlighting recent innovative studies that exemplify how functional assessments of the neuroproteome can provide insight into the mechanisms of drug addiction. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessReview Clinical Proteomics in Colorectal Cancer, a Promising Tool for Improving Personalised Medicine
Received: 30 October 2018 / Revised: 22 November 2018 / Accepted: 29 November 2018 / Published: 2 December 2018
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Abstract
Colorectal cancer is the third most common and the fourth most lethal cancer worldwide. In most of cases, patients are diagnosed at an advanced or even metastatic stage, thus explaining the high mortality. The lack of proper clinical tests and the complicated procedures
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Colorectal cancer is the third most common and the fourth most lethal cancer worldwide. In most of cases, patients are diagnosed at an advanced or even metastatic stage, thus explaining the high mortality. The lack of proper clinical tests and the complicated procedures currently used for detecting this cancer, as well as for predicting the response to treatment and the outcome of a patient’s resistance in guiding clinical practice, are key elements driving the search for biomarkers. In the present overview, the different biomarkers (diagnostic, prognostic, treatment resistance) discovered through proteomics studies in various colorectal cancer study models (blood, stool, biopsies), including the different proteomic techniques used for the discovery of these biomarkers, are reviewed, as well as the various tests used in clinical practice and those currently in clinical phase. These studies define the limits and perspectives related to proteomic biomarker research for personalised medicine in colorectal cancer. Full article
(This article belongs to the Special Issue Clinical Proteomics 2018)
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Open AccessArticle Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins
Received: 16 October 2018 / Revised: 15 November 2018 / Accepted: 18 November 2018 / Published: 28 November 2018
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Abstract
Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to
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Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessFeature PaperArticle Paraquat-Mediated Oxidative Stress in Anopheles gambiae Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response
Received: 15 October 2018 / Revised: 7 November 2018 / Accepted: 9 November 2018 / Published: 12 November 2018
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Abstract
Paraquat is a potent superoxide (O2)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several
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Paraquat is a potent superoxide (O2)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown. Here, we quantified the mosquito midgut proteomic response to a paraquat-laced sugar meal, and found that An. gambiae midguts were enriched in proteins that are indicative of cells under endoplasmic reticulum (ER) stress. We also carried out qRT-PCR analyses for nine prominent thioredoxin (Trx) and glutathione (GSH)-dependent genes in mosquito midguts post P. falciparum blood meal ingestion to evaluate the concordance between transcripts and proteins under different oxidative stress conditions. Our data revealed an absence of significant upregulation in the Trx and GSH-dependent genes following infected blood meal ingestion. These data suggest that the intrinsic tolerance of the mosquito midgut to paraquat-mediated oxidative stress is through an ER stress response. These data indicate that mosquitoes have at least two divergent pathways of managing the oxidative stress that is induced by exogenous compounds, and outline the potential application of paraquat-like drugs to act selectively against malaria parasite development in mosquito midguts, thereby blocking mosquito-to-human transmission. Full article
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Open AccessReview Deep Profiling of the Aggregated Proteome in Alzheimer’s Disease: From Pathology to Disease Mechanisms
Received: 22 September 2018 / Revised: 29 October 2018 / Accepted: 7 November 2018 / Published: 12 November 2018
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Abstract
Hallmarks of Alzheimer’s disease (AD), a progressive neurodegenerative disease causing dementia, include protein aggregates such as amyloid beta plaques and tau neurofibrillary tangles in a patient’s brain. Understanding the complete composition and structure of protein aggregates in AD can shed light on the
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Hallmarks of Alzheimer’s disease (AD), a progressive neurodegenerative disease causing dementia, include protein aggregates such as amyloid beta plaques and tau neurofibrillary tangles in a patient’s brain. Understanding the complete composition and structure of protein aggregates in AD can shed light on the as-yet unidentified underlying mechanisms of AD development and progression. Biochemical isolation of aggregates coupled with mass spectrometry (MS) provides a comprehensive proteomic analysis of aggregates in AD. Dissection of these AD-specific aggregate components, such as U1 small nuclear ribonucleoprotein complex (U1 snRNP), provides novel insights into the deregulation of RNA splicing in the disease. In this review, we summarize the methodologies of laser capture microdissection (LCM) and differential extraction to analyze the aggregated proteomes in AD samples, and discuss the derived novel insights that may contribute to AD pathogenesis. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessArticle A Comparative Quantitative LC-MS/MS Profiling Analysis of Human Pancreatic Adenocarcinoma, Adjacent-Normal Tissue, and Patient-Derived Tumour Xenografts
Received: 16 October 2018 / Revised: 1 November 2018 / Accepted: 2 November 2018 / Published: 6 November 2018
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Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide; it develops in a relatively symptom-free manner, leading to rapid disease progression and metastasis, leading to a 5-year survival rate of less than 5%. A lack of dependable diagnostic markers and rapid
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Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide; it develops in a relatively symptom-free manner, leading to rapid disease progression and metastasis, leading to a 5-year survival rate of less than 5%. A lack of dependable diagnostic markers and rapid development of resistance to conventional therapies are among the problems associated with management of the disease. A better understanding of pancreatic tumour biology and discovery of new potential therapeutic targets are important goals in pancreatic cancer research. This study describes the comparative quantitative LC-MS/MS proteomic analysis of the membrane-enriched proteome of 10 human pancreatic ductal adenocarcinomas, 9 matched adjacent-normal pancreas and patient-derived xenografts (PDXs) in mice (10 at F1 generation and 10 F2). Quantitative label-free LC-MS/MS data analysis identified 129 proteins upregulated, and 109 downregulated, in PDAC, compared to adjacent-normal tissue. In this study, analysing peptide MS/MS data from the xenografts, great care was taken to distinguish species-specific peptides definitively derived from human sequences, or from mice, which could not be distinguished. The human-only peptides from the PDXs are of particular value, since only human tumour cells survive, and stromal cells are replaced during engraftment in the mouse; this list is, therefore, enriched in tumour-associated proteins, some of which might be potential therapeutic or diagnostic targets. Using human-specific sequences, 32 proteins were found to be upregulated, and 113 downregulated in PDX F1 tumours, compared to primary PDAC. Differential expression of CD55 between PDAC and normal pancreas, and expression across PDX generations, was confirmed by Western blotting. These data indicate the value of using PDX models in PDAC research. This study is the first comparative proteomic analysis of PDAC which employs PDX models to identify patient tumour cell-associated proteins, in an effort to find robust targets for therapeutic treatment of PDAC. Full article
(This article belongs to the Special Issue Pharmaceutical Proteomics)
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Open AccessCommunication Inhibiting Arginine Methylation as a Tool to Investigate Cross-Talk with Methylation and Acetylation Post-Translational Modifications in a Glioblastoma Cell Line
Received: 30 August 2018 / Revised: 2 October 2018 / Accepted: 17 October 2018 / Published: 20 October 2018
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Abstract
Glioblastomas (GBM) are the most common grade 4 brain tumours; patients have very poor prognosis with an average survival of 15 months after diagnosis. Novel research lines have begun to explore aberrant protein arginine methylation (ArgMe) as a possible therapeutic target in GBM
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Glioblastomas (GBM) are the most common grade 4 brain tumours; patients have very poor prognosis with an average survival of 15 months after diagnosis. Novel research lines have begun to explore aberrant protein arginine methylation (ArgMe) as a possible therapeutic target in GBM and ArgMe inhibitors are currently in clinical trials. Enzymes known as protein arginine methyltransferases (PRMT1-9) can lead to mono- or di-ArgMe, and in the latter case symmetric or asymmetric dimethylation (SDMA and ADMA, respectively). Using the most common GBM cell line, we have profiled the expression of PRMTs, used ArgMe inhibitors as tools to investigate post-translational modifications cross-talk and measured the effect of ArgMe inhibitors on cell viability. We have identified novel SDMA events upon inhibition of ADMA in GBM cells and spheroids. We have observed cross-talk between ADMA and lysine acetylation in GBM cells and platelets. Treatment of GBM cells with furamidine, a PRMT1 inhibitor, reduces cell viability in 2D and 3D models. These data provide new molecular understanding of a disease with unmet clinical needs. Full article
(This article belongs to the Special Issue Tools for understanding PTM crosstalk)
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Open AccessArticle Identification of Potential Plasma Biomarkers for Abdominal Aortic Aneurysm Using Tandem Mass Tag Quantitative Proteomics
Received: 10 August 2018 / Revised: 11 October 2018 / Accepted: 12 October 2018 / Published: 18 October 2018
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Abstract
Plasma biomarkers that identify abdominal aortic aneurysm (AAA) rupture risk would greatly assist in stratifying patients with small aneurysms. Identification of such biomarkers has hitherto been unsuccessful over a range of studies using different methods. The present study used an alternative proteomic approach
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Plasma biomarkers that identify abdominal aortic aneurysm (AAA) rupture risk would greatly assist in stratifying patients with small aneurysms. Identification of such biomarkers has hitherto been unsuccessful over a range of studies using different methods. The present study used an alternative proteomic approach to find new, potential plasma AAA biomarker candidates. Pre-fractionated plasma samples from twelve patients with AAA and eight matched controls without aneurysm were analyzed by mass spectrometry applying a tandem mass tag (TMT) technique. Eight proteins were differentially regulated in patients compared to controls, including decreased levels of the enzyme bleomycin hydrolase. The down-regulation of this enzyme was confirmed in an extended validation study using an enzyme-linked immunosorbent assay (ELISA). The TMT-based proteomic approach thus identified novel potential plasma biomarkers for AAA. Full article
(This article belongs to the Special Issue Clinical Proteomics 2018)
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Open AccessArticle Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo
Received: 12 September 2018 / Revised: 10 October 2018 / Accepted: 11 October 2018 / Published: 15 October 2018
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Abstract
Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following
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Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessArticle Phosphoproteomic Analysis of the Amygdala Response to Adolescent Glucocorticoid Exposure Reveals G-Protein Coupled Receptor Kinase 2 as a Target for Reducing Motivation for Alcohol
Received: 28 August 2018 / Revised: 28 September 2018 / Accepted: 9 October 2018 / Published: 12 October 2018
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Abstract
Early life stress is associated with risk for developing alcohol use disorders (AUDs) in adulthood. Though the neurobiological mechanisms underlying this vulnerability are not well understood, evidence suggests that aberrant glucocorticoid and noradrenergic system functioning play a role. The present study investigated the
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Early life stress is associated with risk for developing alcohol use disorders (AUDs) in adulthood. Though the neurobiological mechanisms underlying this vulnerability are not well understood, evidence suggests that aberrant glucocorticoid and noradrenergic system functioning play a role. The present study investigated the long-term consequences of chronic exposure to elevated glucocorticoids during adolescence on the risk of increased alcohol-motivated behavior, and on amygdalar function in adulthood. A discovery-based analysis of the amygdalar phosphoproteome using mass spectrometry was employed, to identify changes in function. Adolescent corticosterone (CORT) exposure increased alcohol, but not sucrose, self-administration, and enhanced stress-induced reinstatement with yohimbine in adulthood. Phosphoproteomic analysis indicated that the amygdala phosphoproteome was significantly altered by adolescent CORT exposure, generating a list of potential novel mechanisms involved in the risk of alcohol drinking. In particular, increased phosphorylation at serines 296–299 on the α2A adrenergic receptor (α2AAR), mediated by the G-protein coupled receptor kinase 2 (GRK2), was evident after adolescent CORT exposure. We found that intra-amygdala infusion of a peptidergic GRK2 inhibitor reduced alcohol seeking, as measured by progressive ratio and stress reinstatement tests, and induced by the α2AAR antagonist yohimbine. These results suggest that GRK2 represents a novel target for treating stress-induced motivation for alcohol which may counteract alterations in brain function induced by adolescent stress exposure. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessReview Phosphorylation of the AMPAR-TARP Complex in Synaptic Plasticity
Received: 11 September 2018 / Revised: 4 October 2018 / Accepted: 6 October 2018 / Published: 8 October 2018
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Abstract
Synaptic plasticity has been considered a key mechanism underlying many brain functions including learning, memory, and drug addiction. An increase or decrease in synaptic activity of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complex mediates the phenomena as shown in the cellular models of synaptic
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Synaptic plasticity has been considered a key mechanism underlying many brain functions including learning, memory, and drug addiction. An increase or decrease in synaptic activity of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complex mediates the phenomena as shown in the cellular models of synaptic plasticity, long-term potentiation (LTP), and depression (LTD). In particular, protein phosphorylation shares the spotlight in expressing the synaptic plasticity. This review summarizes the studies on phosphorylation of the AMPAR pore-forming subunits and auxiliary proteins including transmembrane AMPA receptor regulatory proteins (TARPs) and discusses its role in synaptic plasticity. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessReview Exploring Morphine-Triggered PKC-Targets and Their Interaction with Signaling Pathways Leading to Pain via TrkA
Received: 24 August 2018 / Revised: 29 September 2018 / Accepted: 2 October 2018 / Published: 6 October 2018
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Abstract
It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin
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It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessArticle Early Responses to Severe Drought Stress in the Arabidopsis thaliana Cell Suspension Culture Proteome
Received: 13 September 2018 / Revised: 28 September 2018 / Accepted: 30 September 2018 / Published: 2 October 2018
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Abstract
Abiotic stresses are considered the most deleterious factor affecting growth and development of plants worldwide. Such stresses are largely unavoidable and trigger adaptive responses affecting different cellular processes and target different compartments. Shotgun proteomic and mass spectrometry-based approaches offer an opportunity to elucidate
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Abiotic stresses are considered the most deleterious factor affecting growth and development of plants worldwide. Such stresses are largely unavoidable and trigger adaptive responses affecting different cellular processes and target different compartments. Shotgun proteomic and mass spectrometry-based approaches offer an opportunity to elucidate the response of the proteome to abiotic stresses. In this study, the severe drought or water-deficit response in Arabidopsis thaliana was mimicked by treating cell suspension callus with 40% polyethylene glycol for 10 and 30 min. Resulting data demonstrated that 310 proteins were differentially expressed in response to this treatment with a strict ±2.0-fold change. Over-representation was observed in the gene ontology categories of ‘ribosome’ and its related functions as well as ‘oxidative phosphorylation’, indicating both structural and functional drought responses at the cellular level. Proteins in the category ‘endocytosis’ also show significant enrichment and this is consistent with increased active transport and recycling of membrane proteins in response to abiotic stress. This is supported by the particularly pronounced enrichment in proteins of the endosomal sorting complexes that are required for membrane remodelling. Taken together, the findings point to rapid and complex physiological and structural changes essential for survival in response to sudden severe drought stress. Full article
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Open AccessFeature PaperArticle Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A
Received: 18 July 2018 / Revised: 26 September 2018 / Accepted: 27 September 2018 / Published: 29 September 2018
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Abstract
The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity.
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The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA. Full article
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Open AccessArticle Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery
Received: 30 August 2018 / Revised: 17 September 2018 / Accepted: 20 September 2018 / Published: 23 September 2018
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Abstract
The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide
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The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii. Full article
(This article belongs to the Special Issue Neuroproteomics)
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Open AccessArticle Granulocyte-Colony-Stimulating Factor Alters the Proteomic Landscape of the Ventral Tegmental Area
Received: 19 July 2018 / Revised: 17 September 2018 / Accepted: 20 September 2018 / Published: 23 September 2018
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Abstract
Cocaine addiction is characterized by aberrant plasticity of the mesolimbic dopamine circuit, leading to dysregulation of motivation to seek and take drug. Despite the significant toll that cocaine use disorder exacts on society, there are currently no available pharmacotherapies. We have recently identified
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Cocaine addiction is characterized by aberrant plasticity of the mesolimbic dopamine circuit, leading to dysregulation of motivation to seek and take drug. Despite the significant toll that cocaine use disorder exacts on society, there are currently no available pharmacotherapies. We have recently identified granulocyte-colony stimulating factor (G-CSF) as a soluble cytokine that alters the behavioral response to cocaine and which increases dopamine release from the ventral tegmental area (VTA). Despite these known effects on behavior and neurophysiology, the molecular mechanisms by which G-CSF affects brain function are unclear. In this study mice were treated with repeated injections of G-CSF, cocaine or a combination and changes in protein expression in the VTA were examined using an unbiased proteomics approach. Repeated G-CSF treatment resulted in alterations in multiple signaling pathways related to synaptic plasticity and neuronal morphology. While the treatment groups had marked overlap in their effect, injections of cocaine and the combination of cocaine and G-CSF lead to distinct patterns of significantly regulated proteins. These experiments provide valuable information as to the molecular pathways that G-CSF activates in an important limbic brain region and will help to guide further characterization of G-CSF function and evaluation as a possible translational target. Full article
(This article belongs to the Special Issue Neuroproteomics)
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