Chen X, etal., Biochem Biophys Res Commun 2004 Oct 29;323(4):1157-62.
To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptid
e mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis.
Imai A, etal., J Cell Sci. 2004 Apr 15;117(Pt 10):1945-53. Epub 2004 Mar 23.
Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechani
sm underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.
OBJECTIVES: RAB27B is a member of the Rab family GTPases involved in vesicle trafficking, and p53 has recently been implicated in regulating the exosome secretion pathway. Because exosome secretion plays an important role in modulating tumor microenvironment and
invasive growth, we hypothesized that RAB27B and p53 expression might be associated with the aggressive behavior in pancreatic ductal adenocarcinoma, one of the most deadly human malignancies. METHODS: We determined protein expression of RAB27B and p53 in 260 pancreatic tissues (186 malignant and 74 normal or benign) by immunohistochemistry analysis on tissue microarray and their correlation with patients' clinical parameters and overall survival. RESULTS: We found that a high RAB27B protein expression (RAB27B) was significantly associated with perineural and vascular invasion, as well as distant metastasis. Patients with a high RAB27B expression had significantly poorer overall survival in both univariate and multivariate analyses. A significant correlation between RAB27B and p53 expression was observed. CONCLUSIONS: Our data indicate that RAB27B expression is an independent prognostic marker for pancreatic ductal adenocarcinoma and suggest that RAB27B-regulated exosome secretion pathway represents a novel therapeutic target in pancreatic cancer.
The Rab27 subfamily of secretory small GTPase plays a vital role in vesicle trafficking and regulates tumor growth and metastasis in several cancer types. Thus, this research was designed to explore the clinical and prognostic significance of Rab27A and Rab27B in colorectal cancer (CRC) patients. Re
verse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry (IHC) analysis were used to examine Rab27A and Rab27B expression in human CRC cell lines and primary tumors. The correlation of gene expression with clinicopathological features and prognosis was also evaluated. Our results indicated that Rab27A expression was down-regulated in primary tumors compared with matched adjacent tissues (100%, 8/8), as detected by western blot. IHC analysis revealed that the positive expression rate of Rab27A in primary CRC tissues was lower than in adjacent normal tissues (P = 0.005). Negative expression of Rab27A and Rab27B significantly correlated with poor tumor differentiation (both P < 0.001) and positive vascular invasion (P = 0.005, P = 0.021, respectively). Moreover, absence of Rab27A was associated with advanced tumor node metastasis (TNM) stage (P = 0.006), distant metastasis (P = 0.002), and local recurrence (P = 0.038). Survival analysis also showed a significant correlation between unfavorable survival and negative expression of Rab27A (P = 0.002). In addition, positive expression of both Rab27A and Rab27B was a protective factor in CRC. In conclusion, decreased expression of Rab27A and Rab27B, especially Rab27A, closely correlated with tumor progression and are valuable prognostic indicators in CRC patients.
Hou Y, etal., Biochem Biophys Res Commun. 2016 Mar 18;471(4):610-5. doi: 10.1016/j.bbrc.2016.01.180. Epub 2016 Feb 2.
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.
McGrath JA, etal., Am J Hum Genet. 2012 Dec 7;91(6):1115-21. doi: 10.1016/j.ajhg.2012.10.012. Epub 2012 Nov 21.
The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in
three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs(∗)8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and β4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins.
In the present study, in order to analyze abnormalities in DNA methylation in glioma, we utilized a large cohort methylation microarray (119 glioma samples). Genes associated with tumor grade progression were screened through Significance Analysis of Microarrays (SAM) in the methylation microarray.
We found that Rab27b was hypomethylated in high-grade glioma (anaplastic gliomas and glioblastomas) compared with low-grade glioma (astrocytoma, oligodendrocytoma and oligoastrocytoma) (p=0.02). In 52 glioma samples, we determined both the methylation status of the Rab27b promoter region and protein expression, and confirmed a negative correlation between the methylation status and expression (p<0.01). Immunohistochemistry of 91 gliomas revealed that the Rab27b expression scores of high-grade glioma were higher than scores of low-grade gliomas (p<0.01). In highgrade gliomas, patients haboring Rab27b hypomethylation or overexpression had unfavorable survival prognosis. Transwell invasion assays identified that invasive cell number of glioma U87 and LN229 cells decreased when Rab27b was knocked down. Decreased invasion partly resulted from reduced expression and activation of MMP-9 after Rab27b knockdown. Downregulation of Rab27b also suppressed tumor growth in vivo. Hypomethylated Rab27b was identified as a progression-associated and prognostic molecular marker of glioma.
Pu Y, etal., Clin Exp Metastasis. 2016 Apr;33(4):359-72. doi: 10.1007/s10585-016-9783-0. Epub 2016 Feb 25.
MicroRNAs have been identified as key players in the development and progression of osteosarcoma, which is the most common primary malignancy of bone. Sequencing-based miR-omic and quantitative real-time PCR analyses suggested that the expression of miR-193a-3p and miR-193a-5p was decreased by DNA
methylation at their promoter region in a highly metastatic osteosarcoma cell line (MG63.2) relative to their expression in the less metastatic MG63 cell line. Further wound-healing and invasion assays demonstrated that both miR-193a-3p and miR-193a-5p suppressed osteosarcoma cell migration and invasion. Moreover, introducing miR-193a-3p and miR-193a-5p mimics into MG63.2 cells or antagomiRs into MG63 cells confirmed their critical roles in osteosarcoma metastasis. Additionally, bioinformatics prediction along with biochemical assay results clearly suggested that the secretory small GTPase Rab27B and serine racemase (SRR) were direct targets of miR-193a-3p and miR-193a-5p, respectively. These two targets are indeed involved in the miR-193a-3p- and miR-193a-5p-induced suppression of osteosarcoma cell migration and invasion. MiR-193a-3p and miR-193a-5p play important roles in osteosarcoma metastasis through down-regulation of the Rab27B and SRR genes and therefore may serve as useful biomarkers for the diagnosis of osteosarcoma and as potential candidates for the treatment of metastatic osteosarcoma.
Pavlos NJ, etal., J Neurosci. 2010 Oct 6;30(40):13441-53. doi: 10.1523/JNEUROSCI.0907-10.2010.
Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-res
olution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca(2+)-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.
