Application of Schizosaccharomyces japonicus in makgeolli fermentation and its brewing characteristics.

Recently, unconventional yeasts have become popular as fermentation starters in the brewing industry due to the growing consumer demand for aromatic diversity. Specifically, Schizosaccharomyces japonicus has been explored as a potential starter culture for beer and wine production because of its distinct brewing characteristics; however, its application in makgeolli fermentation has not been tested. Therefore, in the present study, two Sz. japonicus strains (SZJ-1 and SZJ-2) were isolated from natural sources, and their brewing characteristics for makgeolli fermentation were compared with those of commercial S. cerevisiae strain. Although the tested isolates showed a lower fermentation and carbon source consumption rate than control-, their overall alcohol fermentation characteristics were suitable for makgeolli production. Regarding flavor composition, Sz. japonicus-fermented makgeolli possessed more ester compounds (e.g., 2-phenylethyl acetate, ethyl acetate, and ethyl decanoate) than S. cerevisiae-fermented makgeolli. Therefore, Sz. japonicus can be used as an alternative culture starter in makgeolli fermentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01265-6.

Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif (228F-S-G-S-G-N-R-L235) is required for ERAD. However, the mechanisms by which the VCP-Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated in vitro and in vivo by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP-Ufd1 interaction and functional ERAD.

Histone deacetylase 6 inhibitor tubastatin A attenuates angiotensin II-induced hypertension by preventing cystathionine γ-lyase protein degradation.

Cystathionine γ-lyase (CSEγ) is a hydrogen sulfide (H2S)-producing enzyme. Endothelial H2S production can mediate vasodilatory effects, contributing to the alleviation of hypertension (high blood pressure). Recent studies have suggested a role of histone deacetylase 6 (HDAC6) in hypertension, although its underlying mechanisms are poorly understood. Here, we addressed the potential regulation of CSEγ by HDAC6 in angiotensin II (AngII)-induced hypertension and its molecular details focusing on CSEγ posttranslational modification. Treatment of mice with a selective HDAC6 inhibitor tubastatin A (TubA) alleviated high blood pressure and vasoconstriction induced by AngII. Cotreatment of the aorta and human aortic endothelial cells with TubA recovered AngII-mediated decreased H2S levels. AngII treatment upregulated HDAC6 mRNA and protein expression, but conversely downregulated CSEγ protein. Notably, potent HDAC6 inhibitors and HDAC6 siRNA as well as a proteasomal inhibitor increased CSEγ protein levels and blocked the downregulatory effect of AngII on CSEγ. In contrast, other HDAC isoforms-specific inhibitors and siRNAs did not show such blocking effects. Transfected CSEγ protein levels were also reciprocally regulated by AngII and TubA, and were reduced by wild-type, but not by deacetylase-deficient, HDAC6. Moreover, TubA significantly increased both protein stability and K73 acetylation level of CSEγ. Consistent with these results, AngII induced CSEγ ubiquitination and degradation, which was inhibited by TubA. Our results indicate that AngII promoted HDAC6-dependent deacetylation of CSEγ at K73 residue, leading to its ubiquitin-mediated proteolysis, which underlies AngII-induced hypertension. Overall, this study suggests that upregulation of CSEγ and H2S through HDAC6 inhibition may be considered as a valid strategy for preventing the progression of hypertension.

NEDD4-induced degradative ubiquitination of phosphatidylinositol 4-phosphate 5-kinase α and its implication in breast cancer cell proliferation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members generate phosphatidylinositol 4,5-bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K-dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin-proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down-regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C-terminal region and ubiquitinated the N-terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)-induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4-mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild-type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα-dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.

Phosphatidylinositol 4-phosphate 5-kinase α contributes to Toll-like receptor 2-mediated immune responses in microglial cells stimulated with lipoteichoic acid.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important lipid regulator of membrane signaling and remodeling processes. Accumulating evidence indicates a link between PIP2 metabolism and Toll-like receptor (TLR) signaling, a key transducer of immune responses such as inflammation, phagocytosis, and autophagy. Microglia are immune effector cells that serve as macrophages in the brain. Here, we examined the potential role of phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα), a PIP2-producing enzyme, in TLR2 signaling in microglial cells. Treatment of BV2 microglial cells with lipoteichoic acid (LTA), a TLR2 agonist, increased PIP5Kα expression in BV2 and primary microglial cells, but not in primary cultures from TLR2-deficient mice. PIP5Kα knockdown of BV2 cells with shRNA significantly suppressed LTA-induced activation of TLR2 downstream signaling, including the production of proinflammatory cytokines and phosphorylation of NF-κB, JNK, and p38 MAP kinase. Such suppression was reversed by complementation of PIP5Kα. PIP5Kα knockdown lowered PIP2 levels and impaired LTA-induced plasma membrane targeting of TIRAP, a PIP2-dependent adaptor required for TLR2 activation. Besides, PIP5Kα knockdown inhibited phagocytic uptake of E. coli particles and autophagy-related vesicle formation triggered by LTA. Taken together, these results support that PIP5Kα can positively mediate TLR2-associated immune responses through PIP2 production in microglial cells.