Identification of the minimal region of peptide derived from ADP-ribosylation factor1 (ARF1) that inhibits IgE-mediated mast cell activation.

Mast cells play a pivotal role in allergic reactions and inflammations. Aggregation of the high affinity IgE receptor (FcεRI) eventually leads to the release of granule components such as histamine, as well as the de novo synthesis of inflammatory cytokines and lipid mediators. These substances are involved in the development of allergy and inflammation. Therefore, efficient inhibitors of mast cell activation would be therapeutically beneficial. We previously demonstrated that the synthetic peptide derived from the NH2-terminal region (2-17: GNIFANLFKGLFGKKE) of a small GTPase ARF1 (ADP-ribosylation factor1) inhibited FcεRI-induced mast cell degranulation. However, detailed structure-activity relationship study of NH2-terminal portion of ARF1 peptide has not been done. In addition, it is still unclear whether the NH2-terminal peptide of ARF1 suppresses FcεRI-induced production of cytokines and lipid mediators such as leukotriene C4 (LTC4) from mast cells. Here we show that amino acid residues K10-K16 are necessary for ARF1 peptide to efficiently inhibit FcεRI-induced activation of bone marrow-derived mast cells (BMMCs), indicated by decreased mast cell degranulation, cytokine secretion and leukotriene release. Furthermore, we show that ARF1 peptide inhibits IgE-mediated passive cutaneous anaphylaxis reaction. Our results suggest that the peptide derived from ARF1 could be developed into a novel anti-allergic agent for therapeutic intervention in allergy and mast cell-related pathologies.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Characterization of two novel ADP ribosylation factors from giant freshwater prawn Macrobrachium rosenbergii and their responses to WSSV challenge.

ADP-ribosylation factors (Arfs) are small GTP-binding proteins that have an essential function in intracellular trafficking and organelle structure. To date, little information is available on the Arfs in the economically important giant freshwater prawn Macrobrachium rosenbergii and their relationship to viral infection. Here we identified two Arf genes from M. rosenbergii (MrArf1 and MrArf2) for the first time. Phylogenetic analysis showed that MrArf1, together with MjArf1 from shrimp Marsupenaeus japonicus belonged to Class I Arfs. By contrast, MrArf2 didn't not match any of the Arfs classes of I/II/III, although it could be clustered with an Arf protein from M. japonicas called MjArfn, which may represent an analog of the Arf. MrArf1 was ubiquitously expressed in all the examined tissues, with the highest transcription level in the hepatopancreas, whereas MrArf2 was only highly expressed in the hepatopancreas and exhibited very low levels in the heart, stomach, gills and intestine. The expression level of MrArf1 in the gills was down-regulated post 24 h WSSV challenge, and reached the maximal level at 48 h. MrArf1 in the hepatopancreas went up from 24 to 48 h WSSV challenge. MrArf2 transcript in the gill also went down at 24 h and then was upregulated at 48 h WSSV challenge. MrArf2 increased significantly in the hepatopancreas 24 h after infection and then went down at 48 h WSSV challenge. RNAi results showed that knockdown of MrArf1 or MrArf2 could inhibit the expression of the envelope protein gene vp28 of the WSSV. So, it could be speculated that MrArf1 and MrArf2 might play important roles in the innate immune system against WSSV infection.

Differences between AGAP1, ASAP1 and Arf GAP1 in substrate recognition: interaction with the N-terminus of Arf1.

The Arf GAPs are a structurally diverse group of proteins that catalyze the hydrolysis of GTP bound to Arf1. Here, we directly compare the role of amino acids 2-17 of Arf1, a GTP- and phospholipid-sensitive switch, for interaction with three Arf GAPs: Arf GAP1, AGAP1 and ASAP1. Sequestration of amino acids 2-17 with an antibody inhibited interaction with the three tested Arf GAPs. Examination of Arf1 mutants also indicated that [2-17]Arf1 is a critical structural determinant of interaction with all three Arf GAPs; however, the effect of specific mutations differed among the GAPs. Compared to wild-type Arf1, Arf1 with the amino terminal 13 ([Delta13]Arf1) and 17 amino acids ([Delta17]Arf1) deleted had 200- and 4000-fold reduced interaction with ASAP1 and 150-fold reduced interaction with AGAP1. In contrast, deletion of the amino terminus of Arf reduced interaction with Arf GAP1 by 5-fold. By analysis of point mutants, we found that lysines 15 and 16 had a greater contribution to productive interaction between Arf1, ASAP1 and AGAP1 than between Arf1 and Arf GAP1. Leucine 8 contributed to the interaction with Arf GAP1 but not with ASAP1 and AGAP1. Amino acids 2-17 of Arf1, isolated from the protein, inhibited GAP activity of Arf GAP1, ASAP1 and AGAP1 and bound directly to ASAP1. Taken together, our results indicate that (i) Arf GAPs interact with amino acids 2-17 of Arf1 and (ii) each subgroup of Arf GAPs has a unique interface with Arf1.

Reevaluation of the effects of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera.

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein-Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atgamma-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 microg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atgamma-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)-Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.