Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome.

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%

Ceruloplasmin-deficient mice show changes in PTM profiles of proteins involved in messenger RNA processing and neuronal projections and synaptic processes.

Ceruloplasmin (Cp) is a multicopper oxidase with ferroxidase properties being of importance to the mobilisation and export of iron from cells and its ability to bind copper. In ageing humans, Cp deficiency is known to result in aceruloplasminemia, which among other is characterised by neurological symptoms. To obtain novel information about the functions of Cp in the central nervous system (CNS) we compared the brain proteome in forebrains from asymptomatic 4-6-month-old Cp-deficient (B6N(Cg)-Cptm1b(KOMP)Wtsi /J) and wild-type mice. Of more than 5600 quantified proteins, 23 proteins, were regulated, whereas more than 1200 proteins had regulated post-translational modifications (PTMs). The genes of the regulated proteins, glycoproteins and phosphoproteins appeared mostly to be located to neurons and oligodendrocyte precursor cells. Cp deficiency especially affected the function of proteins involved in the extension of neuronal projections, synaptic signalling and cellular mRNA processing and affected the expression of proteins involved in neurodegenerative disease and diabetes. Iron concentration and transferrin saturation were reduced in the blood of even younger, 3- to 5-month-old, Cp-deficient mice. Iron act as cofactor in many enzymatic processes and reactions. Changes in iron availability and oxidation as consequence of Cp deficiency could therefore affect the synthesis of proteins and lipids. This proteomic characterisation is to our knowledge the first to document the changes taking place in the CNS-proteome and its phosphorylation and glycosylation state in Cp-deficient mice.

Mass-spectrometric analysis of APOB polymorphism rs1042031 (G/T) and its influence on serum proteome of coronary artery disease patients: genetic-derived proteomics consequences.

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case-control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71-3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45-2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

Quantitative Phosphoproteomics and Acetylomics of Safranal Anticancer Effects in Triple-Negative Breast Cancer Cells.

Safranal, as an aroma in saffron, is one of the cytotoxic compounds in saffron that causes cell death in triple-negative breast cancer cells. Our recent research reported the anti-cancer effects of safranal, which further demonstrated its impact on protein translation, mitochondrial dysfunction, and DNA fragmentation. To better understand the underlying mechanisms, we identified acetylated and phosphorylated peptides in safranal-treated cancer cells. We conducted a comprehensive phosphoproteomics and acetylomics analysis of safranal-treated MDA-MB-231 cells by using a combination of TMT labeling and enrichment methods including titanium dioxide and immunoprecipitation. We provide a wide range of phosphoproteome regulation in different signaling pathways that are disrupted by safranal treatment. Safranal influences the phosphorylation level on proteins involved in DNA replication and repair, translation, and EGFR activation/accumulation, which can lead the cells into apoptosis. Safranal causes DNA damage which is followed by the activation of cell cycle checkpoints for DNA repair. Over time, checkpoints and DNA repair are inhibited and cells are under a mitotic catastrophe. Moreover, safranal prevents repair by the hypo-acetylation of H4 and facilitates the transcription of proapoptotic genes by hyper-acetylation of H3, which push the cells to the brink of death.

A protein-centric view of in vitro biological model systems for schizophrenia.

Schizophrenia (SCZ) is a severe brain disorder, characterized by psychotic, negative, and cognitive symptoms, affecting 1% of the population worldwide. The precise etiology of SCZ is still unknown; however, SCZ has a high heritability and is associated with genetic, environmental, and social risk factors. Even though the genetic contribution is indisputable, the discrepancies between transcriptomics and proteomics in brain tissues are consistently challenging the field to decipher the disease pathology. Here we provide an overview of the state of the art of neuronal two-dimensional and three-dimensional model systems that can be combined with proteomics analyses to decipher specific brain pathology and detection of alternative entry points for drug development.

Integrated proteomics, phosphoproteomics and metabolomics analyses reveal similarities among giant cell granulomas of the jaws with different genetic mutations.

BACKGROUND: Giant cell granuloma of the jaws are benign osteolytic lesions of the jaws. These lesions are genetically characterized by mutually exclusive somatic mutations at TRPV4, KRAS, and FGFR1, and a fourth molecular subgroup which is wild-type for the three mutations. Irrespective of the molecular background, giant cell granulomas show MAPK/ERK activation. However, it remains unclear if these mutations lead to differences in their molecular signaling in giant cell granulomas. METHODS: Metabolomics, proteomics, and phosphoproteomics analyses were carried out in formalin-fixed paraffin-embedded samples of giant cell granuloma of the jaws. The study cohort consisted of five lesions harboring mutations in FGFR1, six in KRAS, five in TRPV4, and five that were wild-type for these mutations. RESULTS: Lesions harboring KRAS or FGFR1 mutations showed overall similar proteomics and metabolomics profiles. In all four groups, metabolic pathways showed similarity in apoptosis, cell signaling, gene expression, cell differentiation, and erythrocyte activity. Lesions harboring TRPV4 mutations showed a greater number of enriched pathways related to tissue architecture. On the other hand, the wild-type group presented increased number of enriched pathways related to protein metabolism compared to the other groups. CONCLUSION: Despite some minor differences, our results revealed an overall similar molecular profile among the groups with different mutational profile at the metabolic, proteic, and phosphopeptidic levels.

Quantitative proteomic study reveals differential expression of matricellular proteins between fibrous dysplasia and cemento-ossifying fibroma pathogenesis.

BACKGROUND: Fibrous dysplasia (FD) and cemento-ossifying fibroma (COF) are the most common gnathic fibro-osseous lesions. These diseases exhibit remarkable overlap of several clinicopathological aspects, and differential diagnosis depends on the combination of histopathological, radiographic, and clinical aspects. Their molecular landscape remains poorly characterized, and herein, we assessed their proteomic and phosphoproteomic profiles. METHODS: The quantitative differences in protein profile of FD and COF were assessed by proteomic and phosphoproteomic analyses of formalin-fixed paraffin-embedded tissue samples. Pathway enrichment analyses with differentially regulated proteins were performed. RESULTS: FD and COF exhibited differential regulation of pathways related to extracellular matrix organization, cell adhesion, and platelet and erythrocytes activities. Additionally, these lesions demonstrated distinct abundance of proteins involved in osteoblastic differentiation and tumorigenesis and differential abundance of phosphorylation of Ser61 of Yes-associated protein 1 (YAP1). CONCLUSIONS: In summary, despite the morphological similarity between these diseases, our results demonstrated that COF and DF present numerous quantitative differences in their proteomic profiles. These findings suggest that these fibro-osseous lesions trigger distinct molecular mechanisms during their pathogenesis. Moreover, some proteins identified in our analysis could serve as potential biomarkers for differential diagnosis of these diseases after further validation.

Depolarization-dependent Induction of Site-specific Changes in Sialylation on N-linked Glycoproteins in Rat Nerve Terminals.

Synaptic transmission leading to release of neurotransmitters in the nervous system is a fast and highly dynamic process. Previously, protein interaction and phosphorylation have been thought to be the main regulators of synaptic transmission. Here we show that sialylation of N-linked glycosylation is a novel potential modulator of neurotransmitter release mechanisms by investigating depolarization-dependent changes of formerly sialylated N-linked glycopeptides. We suggest that negatively charged sialic acids can be modulated, similarly to phosphorylation, by the action of sialyltransferases and sialidases thereby changing local structure and function of membrane glycoproteins. We characterized site-specific alteration in sialylation on N-linked glycoproteins in isolated rat nerve terminals after brief depolarization using quantitative sialiomics. We identified 1965 formerly sialylated N-linked glycosites in synaptic proteins and found that the abundances of 430 glycosites changed after 5 s depolarization. We observed changes on essential synaptic proteins such as synaptic vesicle proteins, ion channels and transporters, neurotransmitter receptors and cell adhesion molecules. This study is to our knowledge the first to describe ultra-fast site-specific modulation of the sialiome after brief stimulation of a biological system.

Characterization of Signaling Pathways Associated with Pancreatic ß-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice.

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic ß-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, ß-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic ß-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, ß-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the ß-cell's adaptive flexibility and indicates that therapeutic approaches applied to encourage ß-cell rest are capable of restoring endogenous ß-cell function. However, mechanisms that regulate ß-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying ß-cell adaptive flexibility in db/db ß-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with ß-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking-core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of ß-cell function, providing improved insight into disease pathogenesis of T2D.

Identification of SRSF10 as a regulator of SMN2 ISS-N1.

Understanding the splicing code can be challenging as several splicing factors bind to many splicing-regulatory elements. The SMN1 and SMN2 silencer element ISS-N1 is the target of the antisense oligonucleotide drug, Spinraza, which is the treatment against spinal muscular atrophy. However, limited knowledge about the nature of the splicing factors that bind to ISS-N1 and inhibit splicing exists. It is likely that the effect of Spinraza comes from blocking binding of these factors, but so far, an unbiased characterization has not been performed and only members of the hnRNP A1/A2 family have been identified by Western blot analysis and nuclear magnetic resonance to bind to this silencer. Employing an MS/MS-based approach and surface plasmon resonance imaging, we show for the first time that splicing factor SRSF10 binds to ISS-N1. Furthermore, using splice-switching oligonucleotides we modulated the splicing of the SRSF10 isoforms generating either the long or the short protein isoform of SRSF10 to regulate endogenous SMN2 exon 7 inclusion. We demonstrate that the isoforms of SRSF10 regulate SMN1 and SMN2 splicing with different strength correlating with the length of their RS domain. Our results suggest that the ratio between the SRSF10 isoforms is important for splicing regulation.

Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP-activated protein kinase and induces breakdown of glycogen.

Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulator of respiration and oxidative phosphorylation. Thus, pharmacological sAC inhibition is likely to affect both cytosolic and mitochondrial sAC and if bioenergetic readouts are studied, the observed effects are likely to stem from inhibition of mitochondrial rather than cytosolic sAC. Here, we report that a pharmacologically induced inhibition of sAC activity lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMP-activated protein kinase (AMPK), and decreases glycogen stores in cultured primary murine astrocytes. From these data and our discussion of the literature, mitochondrial sAC emerges as a key regulator of astrocyte bioenergetics. Lastly, we discuss the challenges of investigating the functional and metabolic roles of cytosolic versus mitochondrial sAC in astrocytes employing the currently available pharmacological tool compounds.

Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling.

Ultraviolet photodissociation (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y, and c/z) are typically observed. Here we provide a detailed investigation of the level of H/D scrambling for all fragment ion types upon UVPD of the peptide scrambling probe P1 (HHHHHHIIKIIK) using an Orbitrap tribrid mass spectrometer equipped with a solid-state 213 nm UV laser. The most abundant UVPD-generated fragment ions (i.e., b/y ions) exhibit extensive H/D scrambling. Similarly, a/x and c/z ions have also undergone H/D scrambling due to UV-induced heating of the precursor ion population. Therefore, dominant b/y ions upon UVPD of protonated peptides are a strong indicator for the occurrence of extensive H/D scrambling of the precursor ion population. In contrast to peptide P1, UV-irradiation of ubiquitin did not induce H/D scrambling in the nonfragmented precursor ion population. However, the UVPD-generated b2 and a4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended.

Integrated Proteomics Reveals Apoptosis-related Mechanisms Associated with Placental Malaria.

Malaria in pregnancy is a public health concern in malaria-endemic areas. Accumulation of maternal immune cells in the placenta and increased levels of inflammatory cytokines caused by sequestration of Plasmodium falciparum-infected erythrocytes have been associated to poor neonatal outcomes, including low birth weight because of fetal growth restriction. Little is known about the molecular changes occurring in a P. falciparum-infected placenta that has developed placental malaria during pregnancy but had the parasites cleared by pharmacological treatment (past infection). We conducted an integrated proteome, phosphoproteome and glycoproteome analysis in past P. falciparum-infected placentas aiming to find molecular changes associated with placental malaria. A total of 2946 proteins, 1733 N-linked glycosites and 4100 phosphosites were identified and quantified in this study, disclosing overrepresented processes related to oxidative stress, protein folding and regulation of apoptosis in past-infected placentas Moreover, AKT and ERK signaling pathways activation, together with clinical data, were further correlated to an increased apoptosis in past-infected placentas. This study showed apoptosis-related mechanisms associated with placental malaria that can be further explored as therapeutic target against adverse pregnancy outcomes.

Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation.

The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ -1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

Divalent Metal Transporter 1 Knock-Down Modulates IL-1ß Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery.

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic ß-cells, consequently cell death. Inhibition of ß-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced ß-cells during IL-1ß exposure. Our findings reveal new phosphosites in the IL-1ß-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1ß exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving ß-cell functions upon exposure to IL-1ß. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in ß-cells after DMT1 silencing.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Characterization of the Molecular Mechanisms Underlying Glucose Stimulated Insulin Secretion from Isolated Pancreatic ß-cells Using Post-translational Modification Specific Proteomics (PTMomics).

Normal pancreatic islet ß-cells (PBCs) abundantly secrete insulin in response to elevated blood glucose levels, in order to maintain an adequate control of energy balance and glucose homeostasis. However, the molecular mechanisms underlying the insulin secretion are unclear. Improving our understanding of glucose-stimulated insulin secretion (GSIS) mechanisms under normal conditions is a prerequisite for developing better interventions against diabetes. Here, we aimed at identifying novel signaling pathways involved in the initial release of insulin from PBCs after glucose stimulation using quantitative strategies for the assessment of phosphorylated proteins and sialylated N-linked (SA) glycoproteins.Islets of Langerhans derived from newborn rats with a subsequent 9-10 days of maturation in vitro were stimulated with 20 mm glucose for 0 min (control), 5 min, 10 min, and 15 min. The isolated islets were subjected to time-resolved quantitative phosphoproteomics and sialiomics using iTRAQ-labeling combined with enrichment of phosphorylated peptides and formerly SA glycopeptides and high-accuracy LC-MS/MS. Using bioinformatics we analyzed the functional signaling pathways during GSIS, including well-known insulin secretion pathways. Furthermore, we identified six novel activated signaling pathways (e.g. agrin interactions and prolactin signaling) at 15 min GSIS, which may increase our understanding of the molecular mechanism underlying GSIS. Moreover, we validated some of the regulated phosphosites by parallel reaction monitoring, which resulted in the validation of eleven new phosphosites significantly regulated on GSIS. Besides protein phosphorylation, alteration in SA glycosylation was observed on several surface proteins on brief GSIS. Interestingly, proteins important for cell-cell interaction, cell movement, cell-ECM interaction and Focal Adhesion (e.g. integrins, semaphorins, and plexins) were found regulated at the level of sialylation, but not in protein expression. Collectively, we believe that this comprehensive Proteomics and PTMomics survey of signaling pathways taking place during brief GSIS of primary PBCs is contributing to understanding the complex signaling underlying GSIS.

Proteomic Analysis of Restored Insulin Production and Trafficking in Obese Diabetic Mouse Pancreatic Islets Following Euglycemia.

For the treatment of patients with prediabetes or diabetes, clinical evidence has emerged that ß-cell function can be restored by glucose-lowering therapeutic strategies. However, little is known about the molecular mechanisms underlying this functional adaptive behavior of the pancreatic ß-cell. This study examines the dynamic changes in protein expression and phosphorylation state associated with (pro)insulin production and secretory pathway function mediated by euglycemia to induce ß-cell rest in obese/diabetic db/db islet ß-cells. Unbiased quantitative profiling of the protein expression and phosphorylation events that occur upon ß-cell adaption during the transition from hyperglycemia to euglycemia was assessed in isolated pancreatic islets from obese diabetic db/db and wild-type (WT) mice using quantitative proteomics and phosphoproteomics together with bioinformatics analysis. Dynamic changes in the expression and phosphorylation of proteins associated with pancreatic ß-cell (pro)insulin production and complementary regulated-secretory pathway regulation were observed in obese diabetic db/db islets in a hyperglycemic environment, relative to WT mouse islets in a normal euglycemic environment, that resolved when isolated db/db islets were exposed to euglycemia for 12 h in vitro. By similarly treating WT islets in parallel, the effects of tissue culture could be mostly eliminated and only those changes associated with resolution by euglycemia were assessed. Among such regulated protein phosphorylation-dependent signaling events were those associated with COPII-coated vesicle-dependent ER exit, ER-to-Golgi trafficking, clathrin-coat disassembly, and a particular association for the luminal Golgi protein kinase, FAM20C, in control of distal secretory pathway trafficking, sorting, and granule biogenesis. Protein expression and especially phosphorylation play key roles in the regulation of (pro)insulin production, correlative secretory pathway trafficking, and the restoration of ß-cell secretory capacity in the adaptive functional ß-cell response to metabolic demand, especially that mediated by glucose.