Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the Nterminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

In yeast, the multisubunit SAGA (Spt-Ada-Gcn5-acetyltransferase) complex acts as a coactivator to recruit the TATA-binding protein (TBP) to the TATA box, a critical step in eukaryotic gene regulation. However, it is unclear which SAGA subunits are responsible for SAGA's direct interactions with TBP and precisely how SAGA recruits TBP to the promoter. We have used chemical crosslinking to identify Spt8 and Ada1 as potential SAGA subunits that interact with TBP, and we find that both Spt8 and SAGA bind directly to TBP monomer in competition with TBP dimer. We further find that Spt8 and SAGA compete with DNA to bind TBP rather than forming a triple complex. Our results suggest a handoff model for SAGA recruitment of TBP: instead of binding together with TBP at the TATA box, activator-recruited SAGA transfers TBP to the TATA box. This simple model can explain SAGA's observed ability to both activate and repress transcription.

The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-␣ production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKK⑀ and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-␣ production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-␣ activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-␣ induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-␣ pathway in response to viral infection. Fas-associated death domain (FADD)5 is an adaptor protein known to be crucial for the mammalian cell extrinsic pathway of apoptosis. Ligand engagement of the tumor necrosis factor receptor (TNF-R) family of death receptors, which include Fas, TNF-R1, and TRAIL-R, leads to recruitment of FADD and caspase-8 to form a death-inducing complex (1-4). For Fas, FADD is directly recruited to the cytoplasmic tail and is part of the membrane-associated Fas⅐FADD⅐caspase-8 complex. In contrast, TNF-R1 activation leads to the assembly of a cytoplasmic complex that consists of either TRADD⅐FADD⅐caspase-8 or RIP1⅐FADD⅐caspase-8 (3). FADD contains two domains that facilitate protein-protein interactions; that is, the death-effector domain (DED) and the death domain (5, 6). The FADD DED participates in self-association and binding to procaspase-8, whereas the death domain interacts with the death receptors, TRADD or RIP1 (5, 7-9).Accumulating evidence also points to a role for FADD in innate immunity. In Drosophila melanogaster, FADD is part of the immune deficiency pathway required for the Drosophila immune defense against the Gram-negative bacteria (10 -12). Immune deficiency, the Drosophila equivalent of mammalian RIP1, interacts with Drosophila FADD and caspase-8 to initiate the NF-B pathway, leading to production of Drosomycin, an anti-bacterial peptide. Drosophila deficient in FADD expression succumb to infection by Gram-negative bacteria (12). In mice, the absence of FADD or caspase-8 prevents TLR-3/4 (Toll-like receptor)-induced B cell proliferation (13,14). In human and mouse fibroblasts, FADD was implicated in the interferon (IFN) pathway ...

Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gcn5 subcomplex of SAGA which acetylates histones H3 and H2B on nucleosomes, and the Piccolo NuA4 complex which acetylates histones H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify E. coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes.

fungal-like particles and macrophage-conditioned medium are inflammatory elicitors for 3T3-L1 adipocytes chanawee Jakkawanpitak, nongporn Hutadilok-towatana & Decha Sermwittayawong ✉ Adipocytes from white-adipose tissue are known to produce inflammatory cytokines, which play a major role in energy balance and metabolism. While they can respond to pathogen-associated molecular pattern (PAMPs) such as lipopolysaccharide (LPS) from bacteria, it is not known whether adipocytes can be stimulated by fungal cells. Previously, adipocytes were shown to produce toll-like receptor 2 (TLR2), a β-glucan receptor, suggesting that they could respond to β-glucan on the fungal cell wall. In this study, we show that heat-killed yeast induce an inflammatory response in adipocytes. Using fungal-like particles, namely laminarin-coated beads (LCB), we find that these particles trigger the expression of many key inflammatory genes in dose-and time-dependent fashions in adipocytes. these results suggest that β-glucan on the fungal cell wall is sufficient to elicit an inflammatory response in adipocytes. In addition, we show that both LCB and LCB-treated conditioned medium from RAW 264.7 murine macrophages (LCB-RM) induce the expression of those inflammatory genes through iKKβ-iκBα proteins. Together, we conclude that the fungal-like particles and the conditioned medium elicit an inflammatory response in adipocytes through the canonical or classical NF-κB pathway.

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Gray oyster mushroom, a common edible mushroom in Thailand, has been used as a source for polysaccharide extraction and purification. In this study, we extracted polysaccharides from the gray oyster mushroom through hot water, ammonium oxalate, and alkaline extraction methods. The extracts were further purified using enzymatic digestion, solvent extraction, and column chromatography. Fourier‐transform infrared spectroscopy reveals that the selected fractions contain β‐(1→3) glucan and mannan. Monosaccharide composition analysis suggests that glucose and mannose are the major constituents of all analyzed fractions. Interestingly, only the gel‐like fraction, namely SG1‐1 could enhance neutrophils’ killing activity on Candida albicans. In addition, the purified fractions stimulated glucose uptake activity in the L6 myotubes in a dose‐dependent manner. Practical applications The ability of those polysaccharides to stimulate glucose uptake in the muscle cells and enhance a neutrophil’s killing activity implicates the antidiabetic and immunostimulatory activities, respectively. Thus, these polysaccharides could be potentially developed into an immunostimulating agent and an alternative medicine for treating diabetes mellitus.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain. K E Y W O R D SGalleria mellonella, MALDI-TOF mass spectrometry, ompA, two-dimensional gel electrophoresis, Vibrio alginolyticus How to cite this article: Bunpa S, Chaichana N, Teng JLL, et al. Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish. J Fish Dis. 2020;43:275-284. https ://doi.