TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.

Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae.

Expression of Cyanobacterial Acyl-ACP Reductase Elevates the Triacylglycerol Level in the Red Alga Cyanidioschyzon merolae.

Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

A MYB-type transcription factor, MYB2, represses light-harvesting protein genes in Cyanidioschyzon merolae.

While searching for transcriptional regulators that respond to changes in light regimes, we identified a MYB domain-containing protein, MYB2, that accumulates under dark and other conditions in the unicellular red alga Cyanidioschyzon merolae. The isolation and analysis of a MYB2 mutant revealed that MYB2 represses the expression of the nuclear-encoded chloroplast RNA polymerase sigma factor gene SIG2, which results in the repression of the chloroplast-encoded phycobilisome genes that are under its control. Since nuclear-encoded phycobilisome and other light-harvesting protein genes are also repressed by MYB2, we conclude that MYB2 has a role in repressing the expression of light-harvesting genes. The MYB2 mutant is sensitive to a prolonged dark incubation, indicating the importance of MYB2 for cell viability in the dark.

TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae.

Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.

[A case of primary congenital glaucoma with unilateral corneal amyloidosis].

BACKGROUND: Histopathological evaluation of primary congenital glaucoma with extensive corneal amyloidosis. CLINICAL FINDINGS: A three-month-old infant. At the first examination, corneal diameter was 12 mm in both eyes, intraocular pressure (IOP) 43 mmHg in the right eye and 47 mmHg in the left eye. A diagnosis was made of primary congenital glaucoma. At 4 months of age bilateral goniotomy, at 1 year and 2 months of age bilateral goniotomy, and at 2 years and 2 months of age bilateral trabeculectomy and the superficial keratectomy of the right eye were done. At 14 years and 11 months of age the enucleation of the right eye was done because of corneal opacity, visual loss, and figure. HISTOPATHOLOGICAL FINDINGS: In the cornea disappearance of Bowman's membrane, accumulation of amyloid in the rough endoplasmic reticulum of the basal cells and subepithelial fibroblasts, and thickeniny of Descemet's membrane could be seen. At the peripheral anterior synechia new Descemet's membrane was spread over the anterior surface of the peripheral iris. CONCLUSION: Unilateral corneal amyloidosis might have been present in infancy, amyloid produced in the rough endoplasmic reticulum of the basal epithelium and subepithelial fibroblasts and deposited in the subepithelial region. At puberty amyloid might have been produced in the subepithelial fibroblasts. The poor IOP control at puberty might have been due to the complication of neovascular glaucoma.

[Histopathologic study on cyclocryotherapy of human and monkey eyes].

PURPOSE: To evaluate the lesion and repair process of the ciliary body and adjacent tissues in glaucomatous human eyes and normal monkey eyes with cyclocryotherapy. METHODS: We used light and electron microscopy to observe the ciliary body and adjacent tissues in five glaucomatous human eyes and five normal monkey eyes which were given cyclocryotherapy. RESULTS: Five absolute glaucomatous eyes were given cyclocryotherapy from one and a half to 3 years before the enucleation. One eye had no coagulated spots on the ciliary process, but five eyes had coagulated spots on the pars plana. The pigment epithelial cells were atrophic or had disappeared, but non-pigment epithelial cells had proliferated by one or two layers. At the lesion of monkey pars plicata cyclocryotherapy up to three months, melanophage phagocytosed pigment epithelium and melanocytes accumulated, although the proliferation of non-pigment epithelial cells was seen. At the lesion of monkey pars plana cyclocryotherapy melanophage accumulation was also marked, although non-pigment epithelial proliferation was greater than in pars plicata cyclocryotherapy. CONCLUSION: As cyclocryotherapy is a blind therapy, it is uncertain and difficult to destroy the ciliary process precisely. The repair process continued up to three months after monkey cyclocryotherapy, but it took up to one year and 6 months after human cyclocryotherapy.