Platelets store self-agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTPRab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B in vitro, but not other GTPases, and enhanced secretion in an in vitro assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4.

Munc13-4 is a Ca2+-dependent membrane- and SNARE-binding protein that promotes membrane fusion.

Upon activation, platelets release many active substances stored in ␣-and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca 2؉ -induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca 2؉ -induced secretion of von Willebrand factor (vWF) stored in ␣-granules, but not that of [ 3 H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 cofractionated with vWF and P-selectin (an ␣-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [ 3 H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing ␣-and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca 2؉ -induced exocytosis of ␣-granules.Certain types of cells contain specialized granules, which are released in response to extracellular stimuli. The process is known as regulated exocytosis, which is often triggered by the increased concentration of intracellular calcium ions, followed by docking and fusion of granule membrane with plasma membrane (1). Platelets contain three types of such granules, densecore granules, ␣-granules, and lysosomes, which are secreted upon activation of platelets (2, 3). Dense-core granules contain small molecules such as 5-hydroxytryptamine (5-HT) 1 and ␣-granules contain newly synthesized proteins such as von Willebrand factors (vWF). Despite the biological significance of these secretions in platelet function (2), the molecular mechanism remains unclear. Recent advances have shed light on the mechanism of intracellular membrane docking/fusion. An ATPase, N-ethylmaleimide-sensitive factor (NSF), and its binding protein, soluble NSF attachment protein (␣-SNAP), are required for priming SNAP -receptors (SNARE) (4) on vesicular (v-) and target (t-) membranes. Then, the cognate v-and t-SNARE (trans) complex on both sides of the membranes are formed, resulting in their docking/fusion (5, 6). Many v-and t-SNARE homologues have been identified from mammals to yeast, and the SNARE complex-mediated mechanism has been demonstrated to play essential roles in many pathways of intracellular vesicle transport (7). In the case of platelets, NSF (8) and ␣-SNAP (8, 9) have recently been shown to be required for the granule secretion. Furthermore, syntaxin2 and synaptosome-associated protein of 23 kDa (SNAP23), which are t-SNARE proteins...

Mutations of the perforin (PRF1) and MUNC13-4 genes distinguish 2 forms of familial hemophagocytic lymphohistiocytosis (FHL2 and FHL3, respectively)

SummaryWeibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Metformin is the first-line drug in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, metformin has an anti-inflammatory function, but the precise mechanism promoting this activity remains unclear. High mobility group box 1 (HMGB1) is an alarmin that is released from necrotic cells and induces inflammatory responses by its cytokine-like activity and is, therefore, a target of anti-inflammatory therapies. Here we identified HMGB1 as a novel metformin-binding protein by affinity purification using a biotinylated metformin analogue. Metformin directly bound to the C-terminal acidic tail of HMGB1. Both in vitro and in vivo, metformin inhibited inflammatory responses induced by full-length HMGB1 but not by HMGB1 lacking the acidic tail. In an acetaminophen-induced acute liver injury model in which HMGB1 released from injured cells exacerbates the initial injury, metformin effectively reduced liver injury and had no additional inhibitory effects when the extracellular HMGB1 was blocked by anti-HMGB1-neutralizing antibody. In summary, we report for the first time that metformin suppresses inflammation by inhibiting the extracellular activity of HMGB1. Because HMGB1 plays a major role in inflammation, our results suggest possible new ways to manage HMGB1-induced inflammation.

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTPRab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca 2؉ concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (␤-␥-imidoguanosine 5-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca 2؉ -induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.In eukaryotic cells, transport between distinct organelles is performed through vesicle trafficking. The final step of vesicle docking/fusion with target membrane is mediated by transsoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 3 complex bridging a vesicle and its target membrane (1). One of the key regulators for the SNARE complex formation is Rab GTPase (2, 3). So far, more than 60 members of Rab GTPases are identified in mammals, and they play critical roles in the specific transport pathways (2-4). Like other GTPases, the activity of Rab is regulated by its GDP/GTP cycle. Rab proteins have GTP-bound active and GDP-bound inactive forms. The activation process is performed by GDP/ GTP exchange mediated by the GDP/GTP exchange factor. GTP-bound Rabs execute their function by interaction with effector proteins. Then, GTP-Rab is inactivated into GDP-Rab by GTP hydrolysis that is mediated by the intrinsic GTPase activity and its enhancer, GTPase-activating protein. Furthermore, the Rab family has a unique regulatory protein named Rab GDP dissociation inhibitor (RabGDI), which extracts GDP-Rab from membrane into cytosol by forming a 1:1 complex and inhibits GDP/GTP exchange (3). RabGDI accompanies Rabs in cytosol to the correct organelles, where they are reactivated by the function of GDI dissociation factor (5-8).Although elucidation of the regulatory mechanism of the GDP/GTP cycle is crucial for understanding the functional mechanism of Rab GTPases, it has not been extensively investigated so far. Small GTPases belonging to Ras and Rho families are predominantly present in the GDP-bound forms under resting conditions and are transiently activated into GTPbound forms upon stimulation (9), indicating that these GTPases function as "switches" that transduce...