Ral GTPase promotes metastasis of pancreatic ductal adenocarcinoma via elevation of TGF-ß1 production.

Pancreatic ductal adenocarcinoma (PDAC), caused by activating mutations in K-Ras, is an aggressive malignancy due to its early invasion and metastasis. Ral GTPases are activated downstream of Ras and play a crucial role in the development and progression of PDAC. However, the underlying mechanisms remain unclear. In this study, we investigated the mechanism of Ral-induced invasion and metastasis of PDAC cells using RalGAPß-deficient PDAC cells with highly activated Ral GTPases. Array analysis and ELISA revealed increased expression and secretion of TGF-ß1 in RalGAPß-deficient PDAC cells compared to control cells. Blockade of TGF-ß1 signaling suppressed RalGAPß deficiency-enhanced migration and invasion in vitro and metastasis in vivo to levels similar to controls. Phosphorylation of c-Jun N-terminal kinase, a repressor of TGF-ß1 expression, was decreased by RalGAPß deficiency. These results indicate that Ral contributes to invasion and metastasis of PDAC cells by elevating autocrine TGF-ß1 signaling at least in part by decreasing c-Jun N-terminal kinase activity.

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Platelet αIIbß3 integrin binds to SARS-CoV-2 spike protein of alpha strain but not wild type and omicron strains.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a pandemic infectious disease, Coronavirus disease 2019 (COVID-19). It causes respiratory infection. Then, it progresses into a systemic infection by involving other organs. This progression mechanism remains to be elucidated, although thrombus formation plays an important role in its progression. Platelets is involved in the thrombus formation by aggregating each other through association of activated αIIbß3 integrin with the Arg-Gly-Asp (RGD) motif-containing its ligands such as fibrinogen and von Willebrand factor. SARS-CoV-2 enters host cells through association of the spike protein (S-protein) with its receptor, angiotensin-converting enzyme 2 (ACE-2), on the host cells. While presence of ACE2 in platelets is suspicious, S-protein harbors the RGD sequences within its receptor binding domain. Therefore, it could be possible SARS-CoV-2 enter platelets through association of S-protein with αIIbß3. In this study, we found that receptor binding domain of S-protein of WT SARS-CoV-2 strain barely bound to isolated healthy human platelets. In contrast, highly toxic alpha-strain-based N501Y substitution strongly bound to platelets in a RGD dependent manner, although binding of S protein did not induce platelet aggregation or activation. This binding may serve for transferring the infection to systemic organs.

Ral GTPase-activating protein regulates the malignancy of pancreatic ductal adenocarcinoma.

The small GTPases RalA and RalB are members of the Ras family and activated downstream of Ras. Ral proteins are found in GTP-bound active and GDP-bound inactive forms. The activation process is executed by guanine nucleotide exchange factors, while inactivation is mediated by GTPase-activating proteins (GAPs). RalGAPs are complexes that consist of a catalytic α1 or α2 subunit together with a common ß subunit. Several reports implicate the importance of Ral in pancreatic ductal adenocarcinoma (PDAC). However, there are few reports on the relationship between levels of RalGAP expression and malignancy in PDAC. We generated RalGAPß-deficient PDAC cells by CRISPR-Cas9 genome editing to investigate how increased Ral activity affects malignant phenotypes of PDAC cells. RalGAPß-deficient PDAC cells exhibited several-fold higher Ral activity relative to control cells. They had a high migratory and invasive capacity. The RalGAPß-deficient cells grew more rapidly than control cells when injected subcutaneously into nude mice. When injected into the spleen, the RalGAPß-deficient cells formed larger splenic tumors with more liver metastases, and unlike controls, they disseminated into the abdominal cavity. These results indicate that RalGAPß deficiency in PDAC cells contributes to high activities of RalA and RalB, leading to enhanced cell migration and invasion in vitro, and tumor growth and metastasis in vivo.

Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells.

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

Downregulation of RalGTPase-activating protein promotes invasion of prostatic epithelial cells and progression from intraepithelial neoplasia to cancer during prostate carcinogenesis.

RalGTPase-activating protein (RalGAP) is an important negative regulator of small GTPases RalA/B that mediates various oncogenic signaling pathways in various cancers. Although the Ral pathway has been implicated in prostate cancer (PCa) development and progression, the significance of RalGAP in PCa has been largely unknown. We examined RalGAPα2 expression using immunohistochemistry on two independent tissue microarray sets. Both datasets demonstrated that the expression of RalGAPα2 was significantly downregulated in PCa tissues compared to adjacent benign prostatic epithelia. Silencing of RalGAPα2 by short hairpin RNA enhanced migration and invasion abilities of benign and malignant prostate epithelial cell lines without affecting cell proliferation. Exogenous expression of wild-type RalGAP, but not the GTPase-activating protein activity-deficient mutant of RalGAP, suppressed migration and invasion of multiple PCa cell lines and was phenocopied by pharmacological inhibition of RalA/B. Loss of Ralgapa2 promoted local microscopic invasion of prostatic intraepithelial neoplasia without affecting tumor growth in a Pten-deficient mouse model for prostate tumorigenesis. Our findings demonstrate the functional significance of RalGAP downregulation to promote invasion ability, which is a property necessary for prostate carcinogenesis. Thus, loss of RalGAP function has a distinct role in promoting progression from prostatic intraepithelial neoplasia to invasive adenocarcinoma.

Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity.

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.

A CD57+ CTL Degranulation Assay Effectively Identifies Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients.

PURPOSE: Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a genetic disorder that results in immune dysregulation. It requires prompt and accurate diagnosis. A natural killer (NK) cell degranulation assay is often used to screen for FHL3 patients. However, we recently encountered two cases of late-onset FHL3 carrying novel UNC13D missense mutations: in these cases, the degranulation assays using freshly isolated and interleukin (IL)-2-activated NK cells yielded contradictory results. Since the defective degranulation of CD57+ cytotoxic T lymphocytes (CTLs) in these cases was helpful for making the diagnosis, we assessed whether the CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell assays. METHODS: Forty additional patients with hemophagocytic lymphohistiocytosis were prospectively screened for FHL3 by measuring the perforin expression in NK cells and the expression of Munc13-4, syntaxin-11, and Munc18-2 in platelets and by performing NK cell and CTL degranulation assays. The results were confirmed by genetic analysis. RESULTS: The freshly isolated NK cell degranulation assay detected FHL3 patients with high sensitivity (100%) but low specificity (71%). The IL-2-stimulated NK cell assay had improved specificity, but 3 out of the 31 non-FHL3 patients still showed degranulation below the threshold level. The CD57+ CTL degranulation assay identified FHL3 patients with high sensitivity and specificity (both 100%). CONCLUSIONS: The CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell-based assays.

Platelets are highly activated in patients of chronic thromboembolic pulmonary hypertension.

OBJECTIVE: Chronic thromboembolic pulmonary hypertension (CTEPH) is a fatal disease that is distinct from pulmonary arterial hypertension (PAH). Although CTEPH is characterized by obstruction of major pulmonary artery because of chronic thrombus, it remains unclear whether CTEPH is associated with prothrombotic condition. APPROACH AND RESULTS: In addition to conventional markers, GTP-bound levels of Rap1, RhoA, RalA, Rac1, and Ras in platelets, which are implicated for platelet activation, were measured in patients without pulmonary hypertension (non-PH, n=15), patients with PAH (n=19), and patients with CTEPH (n=25). Furthermore, the responsiveness to ex vivo thrombin stimulation was also evaluated. The ratios of the P-selectin positive platelets in the non-PH patients, patients with PAH, and patients with CTEPH were 1.40% (median and interquartile range, 0.83-1.82), 2.40% (1.80-3.39), and 2.63% (1.90-8.22), respectively (non-PH versus CTEPH, P<0.01). The activated GPIIb/IIIa-positive platelets were 6.01% (1.34-7.87), 11.39% (5.69-20.86), and 9.74% (7.83-24.01), respectively (non-PH versus CTEPH, P=0.01). GTP-bound RhoA was 1.79% (0.94-2.83), 4.03% (2.01-5.14), and 2.01% (1.22-2.48), respectively (non-PH versus PAH, P=0.04), and GTP-bound RalA was 1.58% (1.08-2.11), 3.02% (2.03-3.54), and 2.64% (1.42-4.28), respectively (non-PH versus PAH, P=0.023; non-PH versus CTEPH, P=0.048). In contrast, Rac1, Rap1, or Ras was not activated in any groups. The platelets of patients with CTEPH exhibited hyperresponsiveness to ex vivo thrombin stimulation compared with those of non-PH patients when evaluated for the surface markers. Either D-dimer or fibrin degradation product level was not increased in patients with CTEPH. CONCLUSIONS: These results provide the first direct evidence that platelets of patients with CTEPH are highly activated and exhibit hyperresponsiveness to thrombin stimulation.

A complete Rab screening reveals novel insights in Weibel-Palade body exocytosis.

Weibel-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.

von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.

Background: Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. Objectives: To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. Methods: VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. Results: We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. Conclusion: VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein.

Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4-deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Flightless-I (Fli-I) regulates the actin assembly activity of diaphanous-related formins (DRFs) Daam1 and mDia1 in cooperation with active Rho GTPase.

Eukaryotic cells dynamically reorganize the actin cytoskeleton to regulate various cellular activities, such as cell shape change, cell motility, cytokinesis, and vesicular transport. Diaphanous-related formins (DRFs), such as Daam1 and mDia1, play central roles in actin dynamics through assembling linear actin filaments. It has been reported that the GTP-bound active Rho binds directly to DRFs and partially unleashes the intramolecular autoinhibition of DRFs. However, whether proteins other than Rho involve the regulation of the actin assembly activity of DRFs has been unclear. Here, we show that Flightless-I (Fli-I), a gelsolin family protein essential for early development, binds directly to Daam1 and mDia1. Fli-I enhances the intrinsic actin assembly activity of Daam1 and mDia1 in vitro and is required for Daam1-induced actin assembly in living cells. Furthermore, Fli-I promotes the GTP-bound active Rho-mediated relief of the autoinhibition of Daam1 and mDia1. Thus, Fli-I is a novel positive regulator of Rho-induced linear actin assembly mediated by DRFs.

Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking.

Ykt6 is an evolutionarily conserved SNARE protein regulating Golgi membrane fusion and other diverse membrane trafficking pathways. Unlike most SNARE proteins, Ykt6 lacks a transmembrane domain but instead has a tandem cysteine motif at the C-terminus. Recently, we have demonstrated that Ykt6 undergoes double prenylation at the C-terminal two cysteines first by farnesyltransferase and then by a newly identified protein prenyltransferase named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of a novel α subunit prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of Rab geranylgeranyltransferase. PTAR1 knockout (KO) cells, where Ykt6 is singly prenylated with a farnesyl moiety, exhibit structural and functional abnormalities in the Golgi apparatus with delayed intra-Golgi trafficking and impaired protein glycosylation. It remains unclear whether the second prenylation of Ykt6 is required for proper trafficking of lysosomal hydrolases from Golgi to lysosomes. Here, we show that lysosomal hydrolases, cathepsin D and ß-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space in PTAR1 KO cells. Moreover, maturation of these hydrolases was disturbed. LC3B, an autophagy marker, was accumulated in PTAR1 KO cells, suggesting defects in cellular degradation pathways. Thus, doubly prenylated Ykt6, but not singly prenylated Ykt6, is critical for the efficient sorting and trafficking of acid hydrolases to lysosomes.

Characterization of the antiplatelet effect of aspirin at enrollment and after 2-year follow-up in a real clinical setting in Japan.

BACKGROUND: Aspirin is an antiplatelet drug widely used for the prevention of cardiovascular diseases. It has been reported that some patients who exhibit a reduced antiplatelet effect of aspirin have higher cardiovascular risk. It is still controversial whether the antiplatelet effect of aspirin diminishes after a few years of treatment. This study aimed to evaluate the antiplatelet effect of aspirin and its 2-year change in Japanese patients. METHODS AND RESULTS: Collagen-induced platelet-aggregability was measured at enrollment by conventional optical aggregometer in 239 patients undergoing antiplatelet therapy with aspirin alone. Among them, 167 patients were evaluated after 2 years. Whole blood aggregability based on the screen-filtration method was also evaluated. Optical aggregometer studies showed that 27% of patients were low-responders. Multivariate analyses revealed that female sex and non-use of calcium-channel blockers were associated with low responsiveness. The antiplatelet effect of aspirin did not decrease after 2 years. Similar data were obtained with the whole blood aggregometer. CONCLUSIONS: In this Japanese patient group, 27% were low-responders to aspirin, and the antiplatelet effect of aspirin did not decrease after a 2-year interval.

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

Down-regulation of RalGTPase-Activating Protein Promotes Colitis-Associated Cancer via NLRP3 Inflammasome Activation.

BACKGROUND & AIMS: Ral guanosine triphosphatase-activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC. METHODS: We used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction. RESULTS: Bone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1ß, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors. CONCLUSIONS: Ral activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.

Asymmetric coiled-coil structure with Guanine nucleotide exchange activity.

Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 A resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 A. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.