Protein Rab3A Research Articles

Rab3a attenuates spinal cord injury by mediating vesicle release

BackgroundRab3a regulates vesicle secretion and transport. Emerging evidences have shown that extracellular vesicles (EVs) can reach target lesions of injured spinal cords and exert a positive effect on these lesions. However, the molecular mechanism by which Rab3a regulates vesicle secretion to ameliorate spinal cord injury (SCI) is not fully understood. MethodsAn SCI rat model was established which was used to examine the pathological changes and Rab3a expression in spinal cord tissue. Rab3a was overexpressed in the model rats to demonstrate its effect on SCI repair. Rab3a was also knocked down in neuronal cells to verify its role in vesicle secretion and neuronal cells. The binding protein of Rab3a was identified by Co-IP and mass spectrometry. ResultsRab3a was significantly downregulated in SCI rats and Rab3a overexpression promoted SCI repair. Rab3a knockdown inhibited the secretion of neuronal cell-derived EVs. Compared to the EVs from the equal number of control neuronal cells, EVs from Rab3a-knockdown neuronal cells promoted M1 macrophage polarization, which in turn, promoted neuronal cell apoptosis. Mechanistically, STXBP1 was identified as a binding protein of Rab3a, and their interaction promoted the secretion of neuronal cell-derived EVs. Furthermore, METTL2b was significantly downregulated in SCI rats, and METTL2b knockdown significantly reduced Rab3a protein expression. ConclusionThese results suggest that Rab3a promotes the secretion of neuronal cell-derived EVs by interacting with its binding protein STXBP1. Neuronal cells-derived EVs inhibited the polarization of M1 macrophages in the spinal cord microenvironment, thereby promoting SCI repair. Our findings provide a theoretical basis for the clinical treatment of SCI.

Developmental coordination disorder: What can we learn from RI mice using motor learning tasks and QTL analysis.

Developmental Coordination Disorder (DCD) is a neurodevelopmental disorder of unknown etiology that affects one in 20 children. There is an indication that DCD has an underlying genetic component due to its high heritability. Therefore, we explored the use of a recombinant inbred family of mice known as the BXD panel to understand the genetic basis of complex traits (i.e., motor learning) through identification of quantitative trait loci (QTLs). The overall aim of this study was to utilize the QTL approach to evaluate the genome-to-phenome correlation in BXD strains of mice in order to better understand the human presentation of DCD. Results of this current study confirm differences in motor learning in selected BXD strains and strains with altered cerebellar volume. Five strains - BXD15, BXD27, BXD28, BXD75, and BXD86 - exhibited the most DCD-like phenotype when compared with other BXD strains of interest. Results indicate that BXD15 and BXD75 struggled primarily with gross motor skills, BXD28 primarily had difficulties with fine motor skills, and BXD27 and BXD86 strains struggled with both fine and gross motor skills. The functional roles of genes within significant QTLs were assessed in relation to DCD-like behavior. Only Rab3a (Ras-related protein Rab-3A) emerged as a high likelihood candidate gene for the horizontal ladder rung task. This gene is associated with brain and skeletal muscle development, but lacked nonsynonymous polymorphisms. This study along with Gill et al. (same issue) is the first studies to specifically examine the genetic linkage of DCD using BXD strains of mice.

Abstract P2045: Regulation Of Atrial Natriuretic Peptide Secretion By Palmitoylation And Modulation Of The Rab3a GTPase Cycle In Cardiac Myocytes

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release in cardiomyocytes. We generated transgenic mice overexpressing the Golgi-localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9 by S-acyl proteomics. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9-mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1 R728A ) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling.

Prediction of male fertility using Ras-related proteins

Identifying effective biomarkers for the diagnosis of male fertility is crucial for improving animal production and treating male infertility in humans. Ras-related proteins (Rab) are associated with morphological and motion kinematic functions in spermatozoa. Moreover, Rab2A, a Rab protein, is a possible male fertility-related biomarker. The present study was designed to identify additional fertility-related biomarkers among the various Rab proteins. First, the expression of Rab proteins (Rab3A, 4, 5, 8A, 9, 14, 25, 27A, and 34A) from 31 duroc boar spermatozoa was measured before and after capacitation; correlation between Rab protein expression and litter size was evaluated by statistical analysis. The results showed that the expression of Rab3A, 4, 5, 8A, 9, and 25 before capacitation and Rab3A, 4, 5, 8A, 9, and 14 after capacitation were negatively correlated with litter size. Moreover, depending on the cut-off values calculated by receiver operating curves, an increase in litter size was observed when evaluating the ability of the Rab proteins to forecast litter size. Therefore, we suggest that Rab proteins may be potential fertility-related biomarkers that could help select superior sires in the livestock industry.

Effects of 7,8-Dihydroxyflavone on Lipid Isoprenoid and Rho Protein Levels in Brains of Aged C57BL/6 Mice

Synaptic impairment may be the main cause of cognitive dysfunction in brain aging that is probably due to a reduction in synaptic contact between the axonal buttons and dendritic spines. Rho proteins including the small GTPase Rac1 have become key regulators of neuronal morphogenesis that supports synaptic plasticity. Small Rho- and Ras-GTPases are post-translationally modified by the isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively. For all GTPases, anchoring in the plasma membrane is essential for their activation by guanine nucleotide exchange factors (GEFs). Rac1-specific GEFs include the protein T lymphoma invasion and metastasis 1 (Tiam1). Tiam1 interacts with the TrkB receptor to mediate the brain-derived neurotrophic factor (BDNF)-induced activation of Rac1, resulting in cytoskeletal rearrangement and changes in cellular morphology. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) acts as a highly affine-selective TrkB receptor agonist and causes the dimerization and autophosphorylation of the TrkB receptor and thus the activation of downstream signaling pathways. In the current study, we investigated the effects of 7,8-DHF on cerebral lipid isoprenoid and Rho protein levels in male C57BL/6 mice aged 3 and 23 months. Aged mice were daily treated with 100 mg/kg b.w. 7,8-DHF by oral gavage for 21 days. FPP, GGPP, and cholesterol levels were determined in brain tissue. In the same tissue, the protein content of Tiam1 and TrkB in was measured. The cellular localization of the small Rho-GTPase Rac1 and small Rab-GTPase Rab3A was studied in total brain homogenates and membrane preparations. We report the novel finding that 7,8-DHF restored levels of the Rho proteins Rac1 and Rab3A in membrane preparations isolated from brains of treated aged mice. The selective TrkB agonist 7,8-DHF did not affect BDNF and TrkB levels, but restored Tiam1 levels that were found to be reduced in brains of aged mice. FPP, GGPP, and cholesterol levels were significantly elevated in brains of aged mice but not changed by 7,8-DHF treatment. Hence, 7,8-DHF may be useful as pharmacological tool to treat age-related cognitive dysfunction although the underlying mechanisms need to be elucidated in detail.

Maternal stress programs accelerated aging of the basal ganglia motor system in offspring

Early-life stress involved in the programming of stress-related illnesses can have a toxic influence on the functioning of the nigrostriatal motor system during aging. We examined the effects of perinatal stress (PRS) on the neurochemical, electrophysiological, histological, neuroimaging, and behavioral correlates of striatal motor function in adult (4 months of age) and old (21 months of age) male rats. Adult PRS offspring rats showed reduced dopamine (DA) release in the striatum associated with reductions in tyrosine hydroxylase-positive (TH+) cells and DA transporter (DAT) levels, with no loss of striatal dopaminergic terminals as assessed by positron emission tomography analysis with fluorine-18-l-dihydroxyphenylalanine. Striatal levels of DA and its metabolites were increased in PRS rats. In contrast, D2 DA receptor signaling was reduced and A2A adenosine receptor signaling was increased in the striatum of adult PRS rats. This indicated enhanced activity of the indirect pathway of the basal ganglia motor circuit. Adult PRS rats also showed poorer performance in the grip strength test and motor learning tasks. The aged PRS rats also showed a persistent reduction in striatal DA release and defective motor skills in the pasta matrix and ladder rung walking tests. In addition, the old rats showed large increases in the levels of SNAP-25 and synaptophysin, which are synaptic vesicle-related proteins in the striatum, and in the PRS group only, reductions in Syntaxin-1 and Rab3a protein levels were observed. Our findings indicated that the age-dependent threshold for motor dysfunction was lowered in PRS rats. This area of research is underdeveloped, and our study suggests that early-life stress can contribute to an increased understanding of how aging diseases are programmed in early-life.

Proteomic profiling of sclerotic hippocampus revealed dysregulated packaging of vesicular neurotransmitters in temporal lobe epilepsy

PurposeTemporal lobe epilepsy (TLE) is the most common type of epilepsy. Hippocampal sclerosis is the most distinctive pathological feature of TLE; however, its role in the pathogenesis of TLE remains to be clarified. We performed global protein expression analysis of hippocampus from TLE patients and controls, aiming to reveal the molecular signaling pathways related to TLE. MethodProteomic and bioinformatic analyses of the hippocampus were performed on 4 TLE and 4 control samples. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), in combination with TMT-6plex quantification, was applied for global protein expression analysis. The proteomics results were validated by Western blot with 25 TLE and 25 control individuals and Immunohistochemistry analysis with 33 TLE and 10 control individuals. ResultsBioinformatics analysis demonstrated differentially expressed proteins in the synaptic vesicle pathway, the prostaglandin synthesis and regulation pathway and endocannabinoids and retrograde modulation of synaptic transmission pathway. Among these, excitatory amino acid transporter 1 (EAAT1) and Vesicular glutamate transporter 1 (VGLUT1) are critical for TLE and dysregulated expression might be closely related to the uptake of extracellular glutamate and contribute to the pathophysiology of TLE. Ras-related protein Rab-3A (RAB3A) downregulation might indicate the TLE-induced compensatory deficit in glutamate release. ConclusionOur study indicates that expression of some proteins involved in the packaging of vesicular neurotransmitters is altered in TLE. In addition, upregulated expression of annexin family proteins, which are also related to TLE, might play an important role in protection against TLE.

The Rab-Rabphilin system in injured human podocytes stressed by glucose overload and angiotensin II.

Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.

Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels

A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD.

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.

Effects of exercise-induced fatigue on the morphology of asymmetric synapse and synaptic protein levels in rat striatum

Corticostriatal synaptic plasticity is considered to be a cellular basis for somatic motor regulation and motor skill learning. Changes in synaptic transmission efficiency underlie functional plasticity, while structural plasticity involves changes in the ultrastructure of the synapse and the levels of synaptic proteins. Exercise-induced fatigue may impair corticostriatal synaptic plasticity, and this impairment may be an important mechanism for exercise-induced fatigue. However, prior research focused mainly on functional plasticity such that the structural plasticity was not well understood. Because corticostriatal synapses are typical asymmetric synapses, here we have used transmission electron microscopy to examine the changes of asymmetry synaptic ultrastructure in rat striatum before and after repetitive exercise-induced fatigue; we have also used western blotting to detect the levels of synaptic active region protein Munc 13, RIM1 and synaptic vesicle protein Rab3A and postsynaptic density PSD-95 protein in rat striatum before and after exercise-induced fatigue. The results showed that the ultrastructure of asymmetry corticostriatal synapses and synaptic protein levels in the striatum of rats were abnormally changed after repetitive exercise-induced fatigue. These abnormal changes in synaptic ultrastructure and related protein levels may be the structural basis for the corticostriatal plasticity impairment after exercise-induced fatigue.

To Pinpoint the Location and the Orientation of Proteins Associated with Dense-Core Vesicles (DCVs) using CLEM

Exocytosis of catecholamine (adrenaline and noradrenaline) from adrenal chromaffin cells is important for stress responses and insulin release from pancreatic beta cells is critical in regulating blood glucose levels. These exocytic processes are highly regulated by a dozens of key proteins in eukaryotic cells. While the origin, transport, and delivery of cargo-loaded vesicles (dense-core vesicles) and the proteins involved is well studied, how these proteins are organized with respect to dense-core vesicles (DCVs) at the nanoscale remains to be explored. Here, we directly image several proteins that are key regulators of the transport, docking, and priming of DCVs at the plasma membrane. Specifically, correlative super-resolution (dSTORM) light and platinum replica transmission electron microscopy (CLEM) imaging was performed on PC12 cells (a rat adrenal chromaffin-derived neuroendocrine cell line) that were transfected with proteins of interest. We aligned super-resolution dSTORM images of Rab proteins (Rab27a and Rab3a) and Rab effector proteins (granuphilin and Rabphilin) with corresponding TEM images of the plasma membrane and observed the precise correlation between proteins and vesicles at the plasma membrane. The nano-scale molecular architecture of DCVs generated from this correlative imaging will help us understand how key proteins guide the docking, priming, and fusion of DCVs in endocrine cells.

Quantitative proteomics of forebrain subcellular fractions in fragile X mental retardation 1 knockout mice following acute treatment with 2-Methyl-6-(phenylethynyl)pyridine: Relevance to developmental study of schizophrenia.

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.

Hippocampus proteomics for effect of Er-xian decoction on ovariectomy induced-learning and memory disabilities in rats

To investigate the effect of Er-xian decoction on ovariectomy induced-learning and memory disability in rats based on the differences in hippocampus proteomic expression, the female rats were ovariectomized for perimenopausal status and then randomly divided into 3 groups, namely sham group, control group and Er-xian decoction group. The rats in sham group and control group were given normal saline by gavage everyday, the rats in Er-xian decoction group were given 12 g·kg⁻¹ of Er-xian decoction by gavage once a day. After treatment for 90 day, all of the animals were tested by Morris water maze, and all of the samples were collected for testing. The hippocampus proteins were detected by Nano-LC-LTQ-Orbitrap system, and identified by Protein Discovery software. The intensity of differential proteins were quantitated by SIEVE software，and the biological functions of protein were analyzed by DIVAD database. The results showed that Er-xian decoction could significantly improve the abilities of learning and memory. Compared with control group， 216 differential proteins with a consistent variation trend with the sham group were found in the Er-xian decoction group. And they mainly concentrated in calcium signaling pathway, Rab3A and Rab14 were key genes with the highest functional classification enrichment score in the gene group. Er-xian decoction could significantly improve the abilities of learning and memory in perimenopausal model rats. The invention effect of Er-xian decoction may be correlated with the calcium signaling pathway and Ras-related protein Rab-3A, and Ras-related protein Rab-14.

ITRAQ-based Proteomic Analysis of APPSw,Ind Mice Provides Insights into the Early Changes in Alzheimer's Disease.

Background: Several proteins have been identified as potential diagnostic biomarkers in imaging, genetic, or proteomic studies in Alzheimer disease (AD) patients and mouse models. However, biomarkers for presymptom diagnosis of AD are still under investigation, as are the presymptom molecular changes in AD pathogenesis.Objective: In this study, we aim to analyzed the early proteomic changes in APPSw,Ind mice and to conduct further functional studies on interesting proteins.Methods: We used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with mass spectrometry to examine the early proteomic changes in hippocampi of APPSw,Ind mice. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immuno-blotting were performed for further validation. Finally, the functions of interesting proteins β-spectrin and Rab3a in APP trafficking and processing were tested by shRNA knockdown, in N2A cells stably expressing β-amyloid precursor protein (APP).Results: The iTRAQ and RT-PCR results revealed the detailed molecular changes in oxidative stress, myelination, astrocyte activation, mTOR signaling and Rab3-dependent APP trafficking in the early stage of AD progression. Knock down of β-spectrin and Rab3a finally led to increased APP fragment production, indicating key roles of β-spectrin and Rab3a in regulating APP processing.Conclusion: Our study provides the first insights into the proteomic changes that occur in the hippocampus in the early stages of the AD mouse model. In addition to improving the understanding of molecular alterations and functional cascades involved in early AD pathogenesis, our findings raise the possibility of developing potential biomarkers and therapeutic targets for early AD.

MiR-124a expression contributes to the monophasic pattern of insulin secretion in islets from pregnant rats submitted to a low-protein diet

To evaluate the role of miR-124a in the regulation of genes involved in insulin exocytosis and its effects on the kinetics of insulin secretion in pancreatic islets from pregnant rats submitted to a low-protein diet. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. Kinetics of the glucose-induced insulin release and measurement of [Ca2+]i in pancreatic islets were assessed by standard protocols. The miR-124a expression and gene transcriptions from pancreatic islets were determined by real-time polymerase chain reaction. In islets from LPP rats, the first phase of insulin release was abrogated. The AUC [Ca2+]i from the LPP group was lower compared with the other groups. miR-124a expression was reduced by a low-protein diet. SNAP-25 mRNA, protein expression, and Rab3A protein content were lower in the LPP rats than in CP rats. Syntaxin 1A and Kir6.2 mRNA levels were decreased in islets from low-protein rats compared with control rats, whereas their protein content was reduced in islets from pregnant rats. Loss of biphasic insulin secretion in islets from LPP rats appears to have resulted from reduced [Ca2+]i due, at least in part, to Kir6.2 underexpression and from the changes in exocytotic elements that are influenced either directly or indirectly by miR-124a.

A Phosphoproteomic Screen Identifies a Guanine Nucleotide Exchange Factor for Rab3A Protein as a Mitogen-activated Protein (MAP) Kinase Phosphatase-5-regulated MAP Kinase Target in Interleukin 6 (IL-6) Secretion and Myogenesis

The mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-regulated MAPK targets in the control of skeletal muscle function and regenerative myogenesis have not been established. To identify MKP-5-regulated MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice. We discovered a novel MKP-5-regulated MAPK substrate called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyperphosphorylated on a phospho-MAPK motif in skeletal muscle of MKP-5-deficient mice. GRAB was found to be phosphorylated by JNK on serine 169. Myoblasts overexpressing a phosphorylation-defective mutant of GRAB containing a mutation at Ser-169 to Ala-169 (GRAB-S169A) inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser-169 was required for the secretion of the promyogenic cytokine interleukin 6 (IL-6). Consistent with this observation, MKP-5-deficient mice exhibited increased circulating IL-6 expression as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP-5-mediated regulation of JNK negatively regulates phosphorylation of GRAB, which subsequently controls secretion of IL-6. These data support the notion that MKP-5 serves as a negative regulator of MAPK-dependent signaling of critical skeletal muscle signaling pathways.

Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis.

BackgroundRab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear.ResultsIn this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared.ConclusionsThe present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies.

3D structure generation, virtual screening and docking of human Ras-associated binding (Rab3A) protein involved in tumourigenesis

Rab3A is expressed predominantly in brain and synaptic vesicles. Rab3A is involved specifically in tethering and docking of synaptic vesicles prior to fusion which is a critical step in regulated release of neurotransmitters. The precise function of Rab3A is still not known. However, up-regulation of Rab3A has been reported in malignant neuroendocrine and breast cancer cells. In the present study, the structure of Rab3A protein was generated using MODELLER 9v8 software. The modeled protein structure was validated and subjected to molecular docking analyses. Docking with GTP was carried out on the binding site of Rab3A using GOLD software. The Rab3A-GTP complex has best GOLD fitness value of 77.73. Ligplot shows hydrogen bondings (S16, S17, V18, G19, K20, T21, S22, S31, T33, A35, S38, T39 and G65) and hydrophobic interacting residues (F25, F32, P34, F36, V37, D62 and A64) with the GTP ligands in the binding site of Rab3A protein. Here, the ligand molecules of NCI diversity set II from the ZINC database against the active site of the Rab3A protein were screened. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. Docking results were analyzed for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and Ligplot was used to measure protein-ligand interactions. Five compounds which possess good inhibitory activity and may act as potential high affinity inhibitors against Rab3A active site were identified. The top ranking molecule (ZINC13152284) has a Glide score of -6.65 kcal/mol, X-Score of -3.02 kcal/mol and GOLD score of 64.54 with 03 hydrogen bonds and 09 hydrophobic contacts. This compound is thus a good starting point for further development of strong inhibitors.

Synapsin II and Rab3a Cooperate in the Regulation of Epileptic and Synaptic Activity in the CA1 Region of the Hippocampus

Some forms of idiopathic epilepsy in animals and humans are associated with deficiency of synapsin, a phosphoprotein that reversibly associates with synaptic vesicles. We have previously shown that the epileptic phenotype seen in synapsin II knock-out mice (SynII(-)) can be rescued by the genetic deletion of the Rab3a protein. Here we have examined the cellular basis for this rescue using whole-cell recordings from CA1 hippocampal pyramidal cells in brain slices. We find that SynII(-) neurons have increased spontaneous activity and a reduced threshold for the induction of epileptiform activity by 4-aminopyridine (4-AP). Using selective recordings of glutamatergic and GABAergic activity we show that in wild-type neurons low concentrations of 4-AP facilitate glutamatergic and GABAergic transmission in a balanced way, whereas in SynII(-) neurons this balance is shifted toward excitation. This imbalance reflects a deficit in inhibitory synaptic transmission that appears to be secondary to reduced Ca(2+) sensitivity in SynII(-) neurons. This suggestion is supported by our finding that synaptic and epileptiform activity at SynII(-) and wild-type synapses is similar when GABAergic transmission is blocked. Deletion of Rab3a results in glutamatergic synapses that have a compromised responsiveness to either low 4-AP concentrations or elevated extracellular Ca(2+). These changes mitigate the overexcitable phenotype observed in SynII(-) neurons. Thus, Rab3a deletion appears to restore the excitatory/inhibitory imbalance observed in SynII(-) hippocampal slices indirectly, not by correcting the deficit in GABAergic synaptic transmission but rather by impairing excitatory glutamatergic synaptic transmission.