Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay.

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS (homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.

ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.

Rab12 localizes to Shiga toxin-induced plasma membrane invaginations and controls toxin transport.

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin-independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin-induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP-Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor-binding B-subunit of Shiga toxin reaching the trans-Golgi/TGN membranes was decreased in Rab12-depleted cells, and that cells were partially protected against intoxication by Shiga-like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady-state localizations of TGN46 and cation-independent mannose-6-phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.

The role of Rab GTPase in Plant development and stress.

Small GTPase is a type of crucial regulator in eukaryotes. It acts as a molecular switch by binding with GTP and GDP in cytoplasm, affecting various cellular processes. Small GTPase were divided into five subfamilies based on sequence, structure and function: Ras, Rho, Rab, Arf/Sar and Ran, with Rab being the largest subfamily. Members of the Rab subfamily play an important role in regulating complex vesicle transport and microtubule system activity. Plant cells are composed of various membrane-bound organelles, and vesicle trafficking is fundamental to the existence of plants. At present, the function of some Rab members, such as RabA1a, RabD2b/c and RabF2, has been well characterized in plants. This review summarizes the role of Rab GTPase in regulating plant tip growth, morphogenesis, fruit ripening and stress response, and briefly describes the regulatory mechanisms involved. It provides a reference for further alleviating environmental stress, improving plant resistance and even improving fruit quality.

Vesicular Trafficking, a Mechanism Controlled by Cascade Activation of Rab Proteins: Focus on Rab27.

Vesicular trafficking is essential for the cell to internalize useful proteins and soluble substances, for cell signaling or for the degradation of pathogenic elements such as bacteria or viruses. This vesicular trafficking also enables the cell to engage in secretory processes for the elimination of waste products or for the emission of intercellular communication vectors such as cytokines, chemokines and extracellular vesicles. Ras-related proteins (Rab) and their effector(s) are of crucial importance in all of these processes, and mutations/alterations to them have serious pathophysiological consequences. This review presents a non-exhaustive overview of the role of the major Rab involved in vesicular trafficking, with particular emphasis on their involvement in the biogenesis and secretion of extracellular vesicles, and on the role of Rab27 in various pathophysiological processes. Therefore, Rab and their effector(s) are central therapeutic targets, given their involvement in vesicular trafficking and their importance for cell physiology.

Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for NTRK fusion cancers.

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (NTRK1, NTRK2 and NTRK3) genes, respectively. TRKs can regulate cell proliferation, differentiation and even apoptosis through the RAS/MAPKs, PI3K/AKT and PLCγ pathways. Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors, and TRKs have become promising antitumor targets. Therefore, achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications. This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins, the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity, and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.

Actions of Rab27B-GTPase on mammalian central excitatory synaptic transmission.

Members of the Rab3 gene family are considered central to membrane trafficking of synaptic vesicles at mammalian central excitatory synapses. Recent evidence, however, indicates that the Rab27B-GTPase, which is highly homologous to the Rab3 family, is also enriched on SV membranes and co-localize with Rab3A and Synaptotagmin at presynaptic terminals. While functional roles of Rab3A have been well-established, little functional information exists on the role of Rab27B in synaptic transmission. Here we report on functional effects of Rab27B at SC-CA1 and DG-MF hippocampal synapses. The data establish distinct functional actions of Rab27B and demonstrate functions of Rab27B that differ between SC-CA1 and DG-MF synapses. Rab27B knockout reduced frequency facilitation compared to wild-type (WT) controls at the DG/MF-CA3 synaptic region, while increasing facilitation at the SC-CA1 synaptic region. Remarkably, Rab27B KO resulted in a complete elimination of LTP at the MF-CA3 synapse with no effect at the SC-CA1 synapse. These actions are similar to those previously reported for Rab3A KO. Specificity of action on LTP to Rab27B was confirmed as LTP was rescued in response to lentiviral infection and expression of human Rab27B, but not to GFP, in the DG in the Rab27B KO mice. Notably, the effect of Rab27B KO on MF-CA3 LTP occurred in spite of continued expression of Rab3A in the Rab27B KO. Overall, the results provide a novel perspective in suggesting that Rab27B and Rab3A act synergistically, perhaps via sequential effector recruitment or signaling for presynaptic LTP expression in this hippocampal synaptic region.

Involvement of ARHGEF10, GEF for RhoA, in Rab6/Rab8-mediating membrane traffic.

Small GTPases play crucial roles in the maintenance of a homeostatic environment and appropriate movements of the cell. In these processes, the direct or indirect interaction between distinct small GTPases could be required for regulating mutual signaling pathways. In our recent study, ARHGEF10, known as a guanine nucleotide exchange factor (GEF) for RhoA, was indicated to interact with Rab6A and Rab8A, which are known to function in the exocytotic pathway, and colocalized with these Rabs at exocytotic vesicles. Moreover, it was suggested that ARHGEF10 is involved in the regulation of Rab6A and Rab8A localization and invasion of breast carcinoma cells, in which Rab8 also acts via regulation of membrane trafficking. These results may reveal the existence of a novel small GTPase cascade which connects the signaling of these Rabs with RhoA during membrane trafficking. In this mini-review, we consider the possible functions of ARHGEF10 and RhoA in the Rab6- and Rab8-mediated membrane trafficking pathway.

Proportional subcellular localization of Arabidopsis thaliana RabA1a.

Subcellular localization of trafficking proteins in a single cell affects the assembly of trafficking machinery between organelles and vesicles throughout the targeting pathway. RabGTPase is one of the regulators to direct specific targeting of cargo molecules depending on GDP/GTP bound status. We have recently determined the crystal structures of GDP-bound inactive and both GTP- and GppNHp-bound active forms of Arabidopsis RabA1a. It is notable that the switch regions of RabA1a exhibit conformational changes derived by GDP or GTP binding. However, it was not clear that where the GDP- or GTP-bound RabA1a is localized at the subcellular level in a cell. Here we demonstrate that the distinct proportion of subcellular localization of RabA1a depends on its site-specific mutation as the GDP- or GTP-bound form. RabA1a proteins located at the plasma membrane, endosomes, and cytosol. While the GDP-bound form of RabA1aS27N located more at endosomes than the plasma membrane compared to the proportions of RabA1a wild-type, and the GTP-bound RabA1aQ72L located mainly at the plasma membrane in comparison to RabA1a wild-type and RabA1aS27N. These distinct proportional localizations of RabA1a enable a cognate interaction between inactive/active RabA1 and effector molecules to direct specific targeting of its cargo molecules.

Interdependency Between Autophagy and Synaptic Vesicle Trafficking: Implications for Dopamine Release.

Autophagy (ATG) and the Ubiquitin Proteasome (UP) are the main clearing systems of eukaryotic cells, in that being ultimately involved in degrading damaged and potentially harmful cytoplasmic substrates. Emerging evidence implicates that, in addition to their classic catalytic function in the cytosol, autophagy and the proteasome act as modulators of neurotransmission, inasmuch as they orchestrate degradation and turnover of synaptic vesicles (SVs) and related proteins. These findings are now defining a novel synaptic scenario, where clearing systems and secretory pathways may be considered as a single system, which senses alterations in quality and distribution (in time, amount and place) of both synaptic proteins and neurotransmitters. In line with this, in the present manuscript we focus on evidence showing that, a dysregulation of secretory and trafficking pathways is quite constant in the presence of an impairment of autophagy-lysosomal machinery, which eventually precipitates synaptic dysfunction. Such a dual effect appears not to be just incidental but it rather represents the natural evolution of archaic cell compartments. While discussing these issues, we pose a special emphasis on the role of autophagy upon dopamine (DA) neurotransmission, which is early affected in several neurological and psychiatric disorders. In detail, we discuss how autophagy is engaged not only in removing potentially dangerous proteins, which can interfere with the mechanisms of DA release, but also the fate of synaptic DA vesicles thus surveilling DA neurotransmission. These concepts contribute to shed light on early mechanisms underlying intersection of autophagy with DA-related synaptic disorders.

Rab-Effector-Kinase Interplay Modulates Intralumenal Fragment Formation during Vacuole Fusion.

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity.

ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.

Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells.

Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.