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.

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.

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.

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.

Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis.

Ribosome biogenesis is essential for maintaining basic cellular activities although its mechanism is not fully understood. Inhibitor of growth 4 (ING4) is a member of ING family while its cellular functions remain controversial. Here, we identified several nucleolar proteins as novel ING4 interacting proteins. ING4 localized in the nucleus with strong accumulation in the nucleolus through its plant homeodomain, which is known to interact with histone trimethylated H3K4, commonly present in the promoter of active genes. ING4 deficient cells exhibited slower proliferation and the alteration in nucleolar structure with reduced rRNA transcription, which was rescued by exogenous expression of GFP-ING4 to the similar levels of wild type cells. In the ING4 deficient cells, histone H3K9 acetylation and the key rRNA transcription factor UBF at the promoter of rDNA were reduced, both of which were also recovered by exogenous GFP-ING4 expression. Thus, ING4 could positively regulate rRNA transcription through modulation of histone modifications at the rDNA promoter.

Impact of anthropogenic activities on water quality and plankton communities in the Day River (Red River Delta, Vietnam).

Planktons are a major component of food web structure in aquatic ecosystems. Their distribution and community structure are driven by the combination and interactions between physical, chemical, and biological factors within the environment. In the present study, water quality and the community structure of phytoplankton and zooplankton were monthly investigated from January to December 2015 at 11 sampling sites along the gradient course of the Day River (Red River Delta, northern Vietnam). The study demonstrated that the Day River was eutrophic with the average values of total phosphorus concentration 0.17 mg/L, total nitrogen concentration 1.98 mg/L, and Chl a 54 µg/L. Microscopic plankton analysis showed that phytoplankton comprised 87 species belonging to seven groups in which Chlorophyceae, Bacillariophyceae, and Cyanobacteria accounted for the most important constituents of the river's phytoplankton assemblage. A total 53 zooplankton species belonging to three main groups including Copepoda, Cladocera, and Rotatoria were identified. Plankton biomass values were greatest in rainy season (3002.10-3 cell/L for phytoplankton and 12.573 individuals/m3 for zooplankton). Using principal correspondence and Pearson correlation analyses, it was found that the Day River was divided into three main site groups based on water quality and characteristics of plankton community. Temperature and nutrients (total phosphorus and total nitrogen) are key factors regulating plankton abundance and distribution in the Day River.

Prostaglandin E2 inhibits neutrophil extracellular trap formation through production of cyclic AMP.

BACKGROUND AND PURPOSE: Upon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear. EXPERIMENTAL APPROACH: The effects of PGE2 , agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model. KEY RESULTS: PGE2 inhibited PMA-induced NET formation in vitro through EP2 and EP4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP2 receptor agonist. CONCLUSIONS AND IMPLICATIONS: PGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.