Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of ß-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

IL-12 improves the anti-HCC efficacy of dendritic cells loaded with exosomes from overexpressing Rab27a tumor cells.

Determining the appropriate source of antigens for optimal antigen presentation to T cells is a major challenge in designing dendritic cell (DC) -based therapeutic strategies against hepatocellular carcinoma (HCC). Tumor-derived exosomes (Tex) express a wide range of tumor antigens, making them a promising source of antigens for DC vaccines. As reported, the exosomes secreted by tumor cells can inhibit the antitumor function of immune cells. In this study, we transfected hepatocellular carcinoma cells with Rab27a to enhance the yield of exosomes, which were characterized using transmission electron microscopy and Western blot analysis. We found that Tex secreted by overexpressing Rab27a Hepatocellular carcinoma cell lines pulsed DC is beneficial for the differentiation and maturation of DCs but inhibits the secretion of the IL-12 cytokine. Consequently, we developed a complementary immunotherapy approach by using Tex as an antigen loaded onto DCs, in combination with the cytokine IL-12 to induce antigen-specific cytotoxic T lymphocytes （CTLs）. The results indicated that the combination of DC-Tex and IL-12 was more effective in stimulating T lymphocyte proliferation, releasing IFN-γ， and enhancing cytotoxicity compared to using exosomes or IL-12 alone. Additionally, the inclusion of IL-12 also compensated for the reduced IL-2 secretion by DCs caused by Tex. Moreover, in a BALB/c nude mice model of hepatocellular carcinoma, CTLs induced by DC-Tex combined with IL-12 maximized the tumor-specific T-cell immune effect and suppressed tumor growth. Thus, Tex provides a novel and promising source of antigens, with cytokines compensating for the shortcomings of Tex as a tumor antigen. This work helps to clarify the role of exosomes in tumor immunotherapy and may offer a safe and effective prospective strategy for the clinical application of exosome-based cellular immunotherapy.

Campothecin suppresses cell proliferation and migration in head and neck squamous cell carcinoma by blocking RAB27A-mediated phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway.

Camptothecin (CPT), a naturally occurring alkaloid derived from the Camptotheca acuminate plant, exerts anti-tumor properties. However, its specific impact on head and neck squamous cell carcinoma (HNSCC) remains uncertain. The study was to explore the action and mechanism of CPT on HNSCC cells. First, two HNSCC cell lines (FaDu and TU686) and a normal immortalized keratinocyte (HEK001) cell line, were exposed to a spectrum of CPT concentrations (ranging from 10 to 50 µM) for durations of 24 h and 48 h. Cell viability, proliferation, migration, and invasion were assessed by CCK-8 assay, EdU incorporation assay, wound healing assay and transwell assay. Subsequently, si-RAB27A or negative control (NC) was introduced into FaDu and TU686 cells through transfection, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was manipulated with L740Y-P, an activator of this pathway. The expression of proliferating cell nuclear antigen (PCNA), E-cadherin, PI3K/AKT signaling factors and RAB27A were determined by Western blot analysis. RAB27A was detected by immunofluorescence assay. It was found that CPT significantly hindered the viability, proliferation (p<0.01), migration (p<0.001), and invasion (p<0.001) of FaDu and TU686 cells. At the molecular level, administration of CPT caused a decline in the expression of PCNA, P-PI3K, P-AKT, and RAB27A, alongside an elevation in E-cadherin levels within HNSCC cells (p<0.05, p<0.01 and p<0.001). Reducing RAB27A expression enhanced the suppressive impacts of CPT on HNSCC cell viability (p<0.05 and p<0.01), migration (p<0.001) and invasion (p<0.01), these effects that were reversed upon treatment with L740Y-P in HNSCC cells (p<0.001). In summary, our study highlights the efficacy of CPT in HNSCC, demonstrating its influence on cell processes via the RAB27A-mediated PI3K/AKT pathway.

Effects of a Global Rab27a Null Mutation on Murine PVAT and Cardiovascular Function.

BACKGROUND: RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. METHODS: Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. RESULTS: Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. CONCLUSIONS: Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection.

BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

GDP-bound Rab27a regulates clathrin disassembly through HSPA8 after insulin secretion.

Clathrin-dependent endocytosis is a key process for secretory cells, in which molecules on the plasma membrane are both degraded and recycled in a stimulus-dependent manner. There are many reports showing that disruption of endocytosis is involved in the onset of various diseases. Recently, it has been reported that such disruption in pancreatic ß-cells causes impaired insulin secretion and might be associated with the pathology of diabetes mellitus. Compared with exocytosis, there are few reports on the molecular mechanism of endocytosis in pancreatic ß-cells. We previously reported that GDP-bound Rab27a regulates endocytosis through its GDP-dependent effectors after insulin secretion. In this study, we identified heat shock protein family A member 8 (HSPA8) as a novel interacting protein for GDP-bound Rab27a. HSPA8 directly bound GDP-bound Rab27a via the ß2 region of its substrate binding domain (SBD). The ß2 fragment was capable of inhibiting the interaction between HSPA8 and GDP-bound Rab27a, and suppressed glucose-induced clathrin-dependent endocytosis in pancreatic ß-cells. The region also affected clathrin dynamics on purified clathrin-coated vesicles (CCVs). These results suggest that the interaction between GDP-bound Rab27a and HSPA8 regulates clathrin disassembly from CCVs and subsequent vesicle transport. The regulatory stages in endocytosis by HSPA8 differ from those for other GDP-bound Rab27a effectors. This study shows that GDP-bound Rab27a dominantly regulates each stage in glucose-induced endocytosis through its specific effectors in pancreatic ß-cells.

Rab27a-mediated exosome secretion in anterior cingulate cortex contributes to colorectal visceral pain in adult mice with neonatal maternal deprivation.

Chronic visceral pain is a common symptom of irritable bowel syndrome (IBS). Exosomes are involved in the development of pain. Rab27a can mediate the release of exosomes. The purpose of this study is to investigate how Rab27a-mediated exosome secretion in the anterior cingulate cortex (ACC) regulates visceral hyperalgesia induced with neonatal maternal deprivation (NMD) in adult mice. The colorectal distension method was adopted to measure visceral pain. The BCA protein assay kit was applied to detect the exosome protein concentration. Western blotting, quantitative PCR, and immunofluorescence technique were adopted to detect the expression of Rab27a and the markers of exosomes. Exosomes extracted from ACC were more in NMD mice than in control (CON) mice. Injection of the exosome-specific inhibitor GW4869 in ACC attenuated colorectal visceral pain of NMD mice. Injection of NMD-derived exosomes produced colorectal visceral pain in CON mice. Rab27a was upregulated in ACC of NMD mice. Rab27a was highly expressed in ACC neurons of NMD mice, rather than astrocytes and microglia. Injection of Rab27a-siRNA reduced the release of exosomes and attenuated the colorectal visceral pain in NMD mice. This study suggested that overexpression of Rab27a increased exosome secretion in ACC neurons, thus contributing to visceral hyperalgesia in NMD mice.NEW & NOTEWORTHY This work demonstrated that the expression of Rab27a in the anterior cingulate cortex was upregulated, which mediated multivesicular bodies trafficking to the plasma membrane and led to the increased release of neuronal exosomes, thus contributing to colorectal visceral pain in neonatal maternal deprivation (NMD) mice. Blocking the release of exosomes or downregulation of Rab27a could alleviate colorectal visceral pain in NMD mice. These data may provide a promising strategy for the treatment of visceral pain in irritable bowel syndrome patients.

[Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells].

OBJECTIVE: To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. METHODS: Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. RESULTS: Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. CONCLUSION: RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

Expression and significant roles of the lncRNA NEAT1/miR-493-5p/Rab27A axis in ulcerative colitis.

BACKGROUND: Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported to play regulatory roles in ulcerative colitis (UC). In this study, we aimed to determine the specific roles and action mechanism of the nuclear paraspeckle assembly transcript 1 (NEAT1) in UC. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the lncRNA NEAT1 and miR-493-5p expression levels in patients with UC and healthy volunteers. We determine the forecast linkage points of NEAT1 and miR-493-5p using Starbase and those of miR-493-5p and Rab27A using TargetScan, and further verified them using a double luciferase gene reporter kit. RT-qPCR and Western blot analysis were used to determine the lncRNA NEAT1, miR-493-5p, and Rab27A expression levels in lipopolysaccharide (LPS)-induced Caco-2 cells. Flow cytometry and cell counting kit-8 were used to assess Caco-2 cell viability. Tumor necrosis factor-α, interleukin (IL)-6, IL-8, and IL-1ß levels were determined via an enzyme-linked immunosorbent assay. RESULTS: Expression levels of NEAT1 were upregulated and those of miR-493-5p were downregualted in 10 ng/mL LPS-treated Caco-2 cells and patients with UC. Dual-luciferase gene reporter assay revealed that miR-493-5p is linked to NEAT1, and Rab27A is a downstream target of miR-493-5p. Overexpression of miR-493-5p inhibited the apoptosis and inflammation in LPS-treated Caco-2 cells. Moreover, downregulation of lncRNA NEAT1 expression also inhibited the apoptosis and inflammation in LPS-treated Caco-2 cells, which was reversed by Rab27A plasmid cotransfection. CONCLUSION: Our results revealed that NEAT1 participates in UC progression by inhibiting miR-493-5p expression.

Lack of Rab27a attenuates foam cell formation and macrophage inflammation in uremic apolipoprotein E knockout mice.

As the most common cardiovascular disease, atherosclerosis (AS), is a leading cause of high mortality in patients with chronic renal failure. Rab27a has been reported to regulate the progression of cardiovascular and renal diseases. Nevertheless, little studies investigated the role and mechanism of Rab27a in uremic-accelerated AS (UAAS). An animal model of UAAS was established in apolipoprotein E knockout (apoE-/-) mice using 5/6 nephrectomy (NX). We conducted in vitro and in vivo functional experiments to explore the role of Rab27a in UAAS, including the presence of oxidized low-density lipoprotein (ox-LDL). Rab27a expression was upregulated in the plaque tissues of NX apoE-/- mice. The knockout of Rab27a (Rab27a-/-) reduced AS-induced artery injury, as manifested by the reductions of plaque area, collagen deposition, inflammation and lipid droplet. Besides, cholesterol efflux was increased, while the expression of lipid metabolism-related proteins and the secretions of pro-inflammatory factors were decreased in ox-LDL-induced NX Rab27a-/- apoE-/- mice group. Further, Rab27a deletion inhibited the activation of nuclear factor κB (NF-κB) pathway. In conclusion, our study indicated that Rab27a deficiency attenuated foam cell formation and macrophage inflammation, depending on the NF-κB pathway activation, to inhibit AS progression in uremic apoE-/- mice. This finding may provide a new targeting strategy for UAAS therapy.

Multiple pathways and independent functional pools in insulin granule exocytosis.

In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic ß cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin-8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in ß cells.

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

Rab27b, a Regulator of Exosome Secretion, Is Associated With Peritoneal Metastases in Gastric Cancer.

BACKGROUND/AIM: Peritoneal metastasis (PM) of gastric cancer (GC) leads to poor clinical outcomes. Tumor-derived exosomes promote metastasis via communication between tumor cells and host cells. In this study, we investigated the effect of Rab27, which is required for exosome secretion, on the PM of GC. MATERIALS AND METHODS: We established a stable knockdown of two Rab27 homologs, Rab27a and Rab27b, in human GC cells (58As9) with a high potential of PM. We examined the level of exosome secretion from Rab27-knockdown 58As9 cells by Western blotting and the ability of Rab27b knockdown to suppress PM in 58As9 cells using a mouse xenograft model. In vitro proliferation and invasion assays were performed in the Rab27b-knockdown cells. Next, Rab27b expression was evaluated in human GC tissues by immunohistochemistry. Finally, we assessed the clinicopathological and prognostic significance of Rab27b expression by RT-qPCR in both our and other TCGA datasets of GC. RESULTS: Rab27a and Rab27b knockdown in 58As9 cells decreased the secretion of exosomes, characterized by the endocytic marker CD63. Rab27b knockdown decreased PM in vivo without affecting the in vitro proliferation or invasion ability of 58As9 cells. In human GC tissues, Rab27b was overexpressed in tumor cells. The overall and recurrence-free survival rates were significantly lower in GC patients with high compared to low Rab27b mRNA expression in our and other TCGA datasets. CONCLUSION: Rab27b expression potentially serves as a poor prognostic biomarker, possibly affecting PM via exosome secretion from GC cells.

RAB27A promotes the proliferation and invasion of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types worldwide. Despite significant advances in prevention and diagnosis, CRC is still one of the leading causes of cancer-related mortality globally. RAB27A, the member of RAB27 family of small GTPases, is the critical protein for intracellular secretion and has been reported to promote tumor progression. However, it is controversial for the role of RAB27A in CRC progression, so we explored the exact function of RAB27A in CRC development in this study. Based on the stable colon cancer cell lines of RAB27A knockdown and ectopic expression, we found that RAB27A knockdown inhibited proliferation and clone formation of SW480 colon cancer cells, whereas ectopic expression of RAB27A in RKO colon cancer cells facilitated cell proliferation and clone formation, indicating that RAB27A is critical for colon cancer cell growth. In addition, our data demonstrated that the migration and invasion of colon cancer cells were suppressed by RAB27A knockdown, but promoted by RAB27A ectopic expression. Therefore, RAB27A is identified as an onco-protein in mediating CRC development, which may be a valuable prognostic indicator and potential therapeutic target for CRC.

RAB27A and RAB27B Expression May Predict Lymph Node Metastasis and Survival in Patients With Gastric Cancer.

BACKGROUND/AIM: RAB27A and RAB27B are involved in exosome secretion. To date, there have been many attempts to elucidate the roles of RAB27A and RAB27B in the prognosis of various cancer types. The association of RAB27A and RAB27B expression with the clinical and pathological features was evaluated in patients with stomach cancer. MATERIALS AND METHODS: A total of 360 patients who had undergone surgery for stomach cancer between January 1999 and December 2007 at Gyeongsang National University were enrolled in the study. Disease-free survival (DFS) and disease-specific survival (DSS) were compared according to immunohistochemistry of tumor samples. RAB27A and RAB27B mRNA and protein were also extracted from four stomach cancer cell lines using quantitative polymerase chain reaction and western blotting. RESULTS: Strong nuclear RAB27A expression in tumor samples was statistically significantly correlated with lymph node metastasis. Cytoplasmic RAB27B expression was related to poor disease-free survival and its combined cytoplasmic and membranous expression was related to disease-specific survival of patients with different histopathological types of stomach cancer. High RAB27A expression and high RAB27B expression was found in four stomach cancer cell blocks. Among the four cell lines, NCI-N87 exhibited the lowest relative mRNA density and HS746T exhibited the highest relative protein density for both RAB27A and RAB27B. CONCLUSION: RAB27A and RAB27B expression may help predict lymph node metastasis and survival of patients with gastric cancer.

Novel RAB27A Variant Associated with Late-Onset Hemophagocytic Lymphohistiocytosis Alters Effector Protein Binding.

Autosomal recessive mutations in RAB27A are associated with Griscelli syndrome type 2 (GS2), characterized by hypopigmentation and development of early-onset, potentially fatal hemophagocytic lymphohistiocytosis (HLH). We describe a 35-year old male who presented with recurrent fever, was diagnosed with Epstein-Barr virus-driven chronic lymphoproliferation, fulfilled clinical HLH criteria, and who carried a novel homozygous RAB27A c.551G > A p.(R184Q) variant. We aimed to evaluate the contribution of the identified RAB27A variant in regard to the clinical phenotype as well as cellular and biochemical function. The patient displayed normal pigmentation as well as RAB27A expression in blood-derived cells. However, patient NK and CD8+ T cell exocytosis was low. Ectopic expression of the RAB27A p.R184Q variant rescued melanosome distribution in mouse Rab27a-deficient melanocytes, but failed to increase exocytosis upon reconstitution of human RAB27A-deficient CD8+ T cells. Mechanistically, the RAB27A p.R184Q variant displayed reduced binding to SLP2A but augmented binding to MUNC13-4, two key effector proteins in immune cells. MUNC13-4 binding was particularly strong to an inactive RAB27A p.T23N/p.R184Q double mutant. RAB27A p.R184Q was expressed and could facilitate melanosome trafficking, but did not support lymphocyte exocytosis. The HLH-associated RAB27A variant increased Munc13-4 binding, potentially representing a novel mode of impairing RAB27A function selectively in hematopoietic cells.

Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse.

We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870-61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.

Rab27a-dependent exosomes protect against cerebral ischemic injury by reducing endothelial oxidative stress and apoptosis.

INTRODUCTION: Multicellular crosstalk within the brain tissue has been suggested to play a critical role in maintaining cerebral vascular homeostasis. Exosomes (EXs) mediated cell-cell communication, but its role in cerebral ischemic injury is largely unknown. Rab27a is one of the major genes controlling EX release. Here, we explored the role of Rab27a in regulating brain EXs secretion, and the effects of Rab27a-mediated EXs on ischemia evoked cerebral vascular disruption and brain injury. METHODS: Cerebral ischemia was induced in Rab27a knockout (Rab27a-/- ) and wide type (WT) mice by transient middle cerebral artery occlusion (tMCAO). Differential gene expression analysis was performed in ischemic brain tissue by using mRNA sequencing. EXs isolated from brain tissue of Rab27a-/- and WT mice (EXWT or EXRab27a-/- ) were pre-administrated into tMCAO operated Rab27a-/- mice or oxygen and glucose deprivation (OGD) treated primary brain vascular endothelial cells (ECs). RESULTS: We demonstrated that Rab27a expression in the peri-infarct area of brain was significantly elevated, which was associated with local elevation in EXs secretion. Rab27a deficiency dramatically decreased the level of EXs in brain tissue of normal and tMCAO-treated mice, and Rab27a-/- mice displayed an increase in infarct volume and NDS, and a decrease in cMVD and CBF following tMCAO. Pre-infusion of EXWT increased the brain EXs levels in the tMCAO operated Rab27a-/- mice, accompanied with an increase in cMVD and CBF, and a decrease in infarct volume, NDS, ROS production, and apoptosis. The effects of EXRab27a-/- infusion were much diminished although in a dose-dependent manner. In OGD-treated ECs, EXRab27a-/- showed less effectivity than EXWT in decreasing ROS overproduction and apoptosis, paralleling with down-regulated expression of NOX2 and cleaved caspase-3. CONCLUSION: Our study demonstrates that Rab27a controls brain EXs secretion and functions, contributing to cerebral vascular protection from ischemic insult by preventing oxidative stress and apoptosis via down-regulating NOX2 and cleaved caspase-3 expression.

Exosomal expression of RAB27A and its related lncRNA Lnc-RNA-RP11-510M2 in lung cancer.

OBJECTIVES: Examine the diagnostic role of serum exosomal RAB27A mRNA in lung cancer and evaluate the relation of LncRNAs to lung cancer in association to RAB27A mRNA in Egyptian population. METHODS: Exosomal RNA-based biomarkers RAB27A mRNA and Lnc-RNA-RP11-510M2.10 were selected based on bioinformatic methods, followed by RT-qPCR validation of their expression in serum of 20 patients with lung cancer, 10 patients with COPD and 10 healthy volunteers. we examined their expression in 10 bronchoalveolar lavage samples and assessed correlation with the serum levels. RESULTS: There was an inverse relationship between expression of serum exosomal RAB27A mRNA and Lnc-RNA-RP11-510M2.10 (r = -0.62, p = .00). Both serum exosomal RAB27A mRNA and Lnc-RNA-RP11-510M2.10 showed a significant positive and negative association with lung cancer patients respectively in comparison to patients with COPD and healthy persons (p < .001). CONCLUSION: RAB27A mRNA and Lnc-RNA-RP11-510M2.10 could be used as diagnostic and prognostic biomarker tools for lung cancer.

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.