Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera.

Rab3, a member of the Rab GTPase family, has been found to be involved in innate immunity. However, the precise function of this GTPase in innate immunity remains unknown. In this study, we identified a Rab3 gene (Ha-Rab3) from the cotton bollworm, Helicoverpa armigera and studied its roles in innate immune responses. Expression of Ha-Rab3 was upregulated in the hemocytes of H. armigera larvae after the injection of Escherichia coli or chromatography beads. The dsRNA-mediated knockdown of Ha-Rab3 gene in H. armigera larval hemocytes led to significant reduction in the phagocytosis and nodulation activities of hemocytes against E. coli, significant increase in the bacterial load in larval hemolymph, and significant reduction in the encapsulation activities of hemocytes toward invading chromatography beads. Furthermore, Ha-Rab3 knockdown significantly suppressed spreading of plasmatocytes. These results suggest that Ha-Rab3 plays important roles in H. armigera cellular immune responses, possibly by mediating spreading of hemocytes.

Up-regulation of JAM-1 in AR42J cells treated with activin A and betacellulin and the diabetic regenerating islets.

Pancreatic AR42J cells demonstrate the pluripotency in precursor cells of the gut endoderm and also provide an excellent model system to study the differentiation of the pancreas. Using the mRNA differential display technique, we identified junctional adhesion molecule-1 (JAM-1), a component of the tight junction, was highly up-regulated during the differentiation of AR42J cells, although junctions were not formed. The expression level of JAM-1 showed an up-regulation in the mRNA level after 3 hours and in the protein level after 24 hours in [activin A + betacellulin]-treated AR42J cells. The expressions of its signaling molecules, PAR-3 and atypical PKC lambda, also increased after the addition of activin A + betacellulin. When JAM-1 was over-expressed in [activin A + betacellulin]-treated AR42J cells, tagged-JAM-1 was observed in cytoplasm as vesicular structures and JAM-1 was colocalized with Rab3B and Rab13, members of the Rab family expressed at tight junctions. In streptozotocin-induced regenerating islets, the expression of JAM-1 was also up-regulated in the mRNA level and the protein level. JAM-1 might therefore play an important role in the differentiation of AR42J cells and the regeneration of pancreatic islets.

Significance of immunohistochemical expression of Rab3B and SNAP-25 in growth hormone-producing pituitary adenomas.

Exocytosis proteins play an important role in the secretory activities of anterior pituitary cells and adenoma cells. An immunohistochemical study was conducted to elucidate the functional significance of these proteins in growth hormone (GH)-producing adenomas. We studied 40 GH-producing adenomas, 10 prolactinomas, and 10 clinically nonfunctioning (NF) adenomas immunohistochemically with antibodies specific for Rab3B and SNAP-25, both of which are considered essential in secretory activities of the pituitary, and keratin 8 (clone CAM5.2). The tumor volume (TV) was estimated with high-resolution magnetic resonance imaging. Immunoreactivity for Rab3B, which was granular in the cytoplasm, varied in GH adenomas, but was negative in prolactinomas and NF adenomas. Reactivity for SNAP-25, which was linear on the plasma membrane, varied in GH adenomas, and was intensely positive in prolactinomas, and negative in NF adenomas. In GH adenomas, an increased percentage of adenoma cells with dot-like immunoreactivity for keratin 8 was associated with decreased reactivities for Rab3B (R=0.739, P<0.0001) and SNAP-25 (R=0.840, P<0.0001). Increased reactivity for SNAP-25 correlated positively with plasma GH level per unit TV (R=0.685, P<0.0001). The immunoreactivities for Rab3B and SNAP-25 may reflect the number of secretory granules and exocytosis activity, respectively, in pituitary adenomas, including GH adenomas.

Molecular cloning of the mouse homologue of Rab3c.

Small GTP-binding proteins of the Rab subfamily are key regulators of intracellular vesicle transport. Here we report the isolation of a cDNA clone encoding the complete Rab3c isoform from mouse embryo using a degenerative PCR-based approach. Multiple sequence alignment revealed that the predicted amino acid sequence was identical to the previously identified rat Rab3c isoform and 98% identical to the published bovine Rab3c GTPase from brain. Furthermore by in situ hybridisation, Rab3c mRNA was detectable within various regions of the brain, cartilage and highly enriched within intestinal villi of foetal tissues. Chondrocytes in the hypertrophic zone, but not reserve or proliferative zones, expressed high levels of Rab3c. This pattern of expression corresponds with the genesis of matrix vesicles during endochondral ossification. In all, our results suggest that in addition to its functional role during regulated secretion in brain, Rab3c may play a part in matrix vesicle trafficking during skeletal development.

Subcellular distribution and function of Rab3A-D in pancreatic acinar AR42J cells.

Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J cells.

Syntaxin, VAMP, and Rab3 are selectively expressed during sea urchin embryogenesis.

SNARE and rab protein family members were originally identified in terminally differentiated cell types. These proteins are phylogenetically conserved and while compelling evidence demonstrates their involvement in the secretory pathway, their exact function is debated. We recently identified SNARE protein family members in the sea urchin egg and provided evidence that rab3 functions in the exocytosis of cortical granules. Here we tested the hypothesis that these same proteins might also be present throughout embryogenesis to mediate membrane fusion events. We provide evidence that the sea urchin possesses a low complexity of gene family members of syntaxin, VAMP, and rab3 and that these proteins are not only present during development, but are enriched in regions of the embryo with active secretory roles. We found accumulation of each family member in the apical and basal aspects of cleaving blastomeres, indicative of bidirectional secretion into the extraembryonic environment and blastocoel. Elevated levels of syntaxin, VAMP, and rab3 were also found in the mesodermally derived pigment cells that invade and move within the ectoderm. These cells likely rely on SNARE and rab proteins to enable mobility by mediating the secretion of enzymes that break adhesion to neighboring cells and the extracellular matrix. In addition, these secretory proteins are enriched in the gut following gastrulation. Thus, we conclude that VAMP, syntaxin, and rab3 mediate a variety of secretory events that is important for development.

Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase-activating protein.

We recently purified and characterized from rat brain a GTPase-activating protein (GAP) specific for the Rab3 small G protein subfamily implicated in Ca2+-dependent exocytosis. Rab3 GAP showed two bands with Mr of about 130,000 (p130) and 150,000 (p150) on SDS-polyacrylamide gel electrophoresis. p130, but not p150, showed the catalytic activity. Because p150 was likely the subunit of Rab3 GAP, here we cloned the cDNA of p150, determined its primary structure, and characterized it. The tissue and subcellular distribution patterns of p150 and p130 were similar, and both the proteins were enriched in the synaptic soluble fraction. p150 was co-immunoprecipitated with p130 from this fraction. Recombinant p150 formed a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p150 neither showed the Rab3A GAP activity nor affected the activity of recombinant p130. When p150 and p130 were co-expressed in the cells, the subcellular localization of each protein did not change. These results indicate that p150 is the noncatalytic subunit of Rab3 GAP.