Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

Weibel Palade Bodies: Unique Secretory Organelles of Endothelial Cells that Control Blood Vessel Homeostasis

Vascular endothelial cells produce and release compounds regulating vascular tone, blood vessel growth and differentiation, plasma composition, coagulation and fibrinolysis, and also engage in interactions with blood cells thereby controlling hemostasis and acute inflammatory reactions. These interactions have to be tightly regulated to guarantee smooth blood flow in normal physiology, but also allow specific and often local responses to blood vessel injury and infectious or inflammatory insults. To cope with these challenges, endothelial cells have the remarkable capability of rapidly changing their surface properties from non-adhesive (supporting unrestricted blood flow) to adhesive (capturing circulating blood cells). This is brought about by the evoked secretion of major adhesion receptors for platelets (von-Willebrand factor, VWF) and leukocytes (P-selectin) which are stored in a ready-to-be-used form in specialized secretory granules, the Weibel-Palade bodies (WPB). WPB are unique, lysosome related organelles that form at the trans-Golgi network and further mature by receiving material from the endolysosomal system. Failure to produce correctly matured VWF and release it through regulated WPB exocytosis results in pathologies, most importantly von-Willebrand disease, the most common inherited blood clotting disorder. The biogenesis of WPB, their intracellular motility and their fusion with the plasma membrane are regulated by a complex interplay of proteins and lipids, involving Rab proteins and their effectors, cytoskeletal components as well as membrane tethering and fusion machineries. This review will discuss aspects of WPB biogenesis, trafficking and exocytosis focussing on recent findings describing factors contributing to WPB maturation, WPB-actin interactions and WPB-plasma membrane tethering and fusion.

FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein trafficking pathway

The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors to enter plant cells and suppress host defense responses. Only few of them are conserved across different strains and species. Such ‘core effectors’ may target hub immunity pathways that are essential during Phytophthora pathogens interacting with their hosts. However, the underlying mechanisms of core RXLRs-mediated host immunity manipulation are largely unknown. Here, we report the functional characterization of a P. capsici RXLR effector, RXLR242. RXLR242 expression is highly induced during the infection process. Its ectopic expression in Nicotiana benthamiana promotes Phytophthora infection. RXLR242 physically interacts with a group of RAB proteins, which belong to the small GTPase family and function in specifying transport pathways in the intracellular membrane trafficking system. RXLR242 impedes the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast by interfering the formation of RABE1-7-labeled vesicles. Further analysis indicated that such phenomenon is resulted from competitive binding of RXLR242 to RABE1-7. RXLR242 also interferes trafficking of the membrane-located receptor FLAGELLIN-SENSING 2 (FLS2) through competitively interacting with RABA4-3. Taken together, our work demonstrates that RXLR242 manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein transporting pathway in plant hosts.

Vesicular trafficking is a key determinant of the statin response in acute myeloid leukemia

Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

RabA2b Overexpression Alters the Plasma-Membrane Proteome and Improves Drought Tolerance in Arabidopsis

Rab proteins are small GTPases that are important in the regulation of vesicle trafficking. Through data mining, we identified RabA2b to be stress responsive, though little is known about the involvement of RabA in plant responses to abiotic stresses. Analysis of the RabA2b native promoter showed strong activity during osmotic stress, which required the stress hormone Abscisic acid (ABA) and was restricted to the vasculature. Sequence analysis of the promoter region identified predicted binding motifs for several ABA-responsive transcription factors. We cloned RabA2b and overexpressed it in Arabidopsis. The resulting transgenic plants were strikingly drought resistant. The reduced water loss observed in detached leaves of the transgenic plants could not be explained by stomatal aperture or density, which was similar in all the genotypes. Subcellular localization studies detected strong colocalization between RabA2b and the plasma membrane (PM) marker PIP2. Further studies of the PM showed, for the first time, a distinguished alteration in the PM proteome as a result of RabA2b overexpression. Proteomic analysis of isolated PM fractions showed enrichment of stress-coping proteins as well as cell wall/cuticle modifiers in the transgenic lines. Finally, the cuticle permeability of transgenic leaves was significantly reduced compared to the wild type, suggesting that it plays a role in its drought resistant properties. Overall, these data provide new insights into the roles and modes of action of RabA2b during water stresses, and indicate that increased RabA2b mediated PM trafficking can affect the PM proteome and increase drought tolerance.

AMPK regulates germline stem cell integrity and quiescence through a mir-1/tbc-7/rab-7 pathway in C. elegans.

During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as "dauer". During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that lack AMPK signalling, the germ cells fail to arrest, undergo uncontrolled proliferation and lose their reproductive capacity. These germline defects are accompanied by an altered chromatin landscape and gene expression program. We identified an allele of tbc-7, a RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is regulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. A singular small RNA, mir-1, was identified as a direct negative regulator of tbc-7 expression through the analysis of seed sequences on the 3′UTR of tbc-7. Animals lacking mir-1 are post-dauer sterile, displaying a similar phenotype to AMPK mutants. Altogether, our findings describe a novel mir-1/tbc-7/rab-7 pathway occurring in the neurons that regulates the germ line in a cell non-autonomous manner.

The Regulation of Rab GTPases by Phosphorylation

Rab proteins are small GTPases that act as molecular switches for intracellular vesicle trafficking. Although their function is mainly regulated by regulatory proteins such as GTPase-activating proteins and guanine nucleotide exchange factors, recent studies have shown that some Rab proteins are physiologically phosphorylated in the switch II region by Rab kinases. As the switch II region of Rab proteins undergoes a conformational change depending on the bound nucleotide, it plays an essential role in their function as a ‘switch’. Initially, the phosphorylation of Rab proteins in the switch II region was shown to inhibit the association with regulatory proteins. However, recent studies suggest that it also regulates the binding of Rab proteins to effector proteins, determining which pathways to regulate. These findings suggest that the regulation of the Rab function may be more dynamically regulated by phosphorylation than just through the association with regulatory proteins. In this review, we summarize the recent findings and discuss the physiological and pathological roles of Rab phosphorylation.