SGT2 and MDY2 interact with molecular chaperone YDJ1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Sgt2 was thought to be the homologue of vertebrate SGT (small glutamine tetratricopeptide repeat-containing protein). SGT has been known to interact with both Hsp70 and Hsp90. However, it was not clear whether Sgt2 might have a similar capacity. Here, we showed that Ssa1/Ssa2 (yeast heat shock cognate [Hsc]70), Hsc82 (yeast Hsp90), and Hsp104 coprecipitated with Sgt2 from yeast lysates. Another molecular chaperone, Ydj1, known to interact with Ssal and Hsc82, also coprecipitated with Sgt2. Synthetic lethality between SGT2 and YDJ1 was observed after the cells were under stress, although Sgt2 might not interact physically with Ydj1. We also found that Mdy2 interacted with the N-terminal region of Sgt2 and that Mdy2 appeared to interact physically with Ydj1. Mdy2 therefore may mediate the association of Ydj1 and Sgt2. In addition, the mating efficiency of mdy2delta, sgt2delta, and mdy2deltasgt2delta strains was reduced to a similar extent. Compared with mdy2delta and ydj1delta cells, ydj1deltamdy2delta cells, however, showed a further suppression in mating efficiency. Moreover, MDY2 interacted genetically with YDJ1. These results suggest that protein complexes containing Sgt2 and Mdy2 bring molecular chaperones together to carry out certain chaperoning functions.     

Chaperone-like protein p32 regulates ULK1 stability and autophagy

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32–ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis.Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics.¹ It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations.^{2, 3, 4, 5} Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues.^{6, 7, 8, 9} Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis.¹ Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation.^{10, 11} Notably, p32 was found to form protein complex with a variety of molecules^{7, 12, 13} and has been suggested that it may act as a multifunctional chaperone protein.^{12, 13, 14}ULK1 has a crucial role in mitophagy induction.¹⁵ Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation^{16, 17} and phosphorylation^{18, 19, 20} have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy.²¹ Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a defect in autophagy in EBSS-starved cells, and impairs clearance of dysfunctional mitochondria in cells exposed to mitochondrial uncoupler. Importantly, these autophagy and mitophagy defects can be restored by re-introducing ULK1 into p32-deficient cells, demonstrating ULK1 functions as a crucial downstream effector of p32.     

A forward loop between glioma and microglia: glioma-derived extracellular matrix-activated microglia secrete IL-18 to enhance the migration of glioma cells

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor-related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here, we found that C6 glioma--but not primary astrocyte-derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV-2 cell line, and activated microglia-secreted interleukin (IL)-18, a potent inflammatory cytokine of the IL-1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL-18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL-18-induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase inhibitor, and protein kinase G inhibitor. Taken together, these results indicate that IL-18 secreted by microglia, which was activated by C6 glioma-derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway.     

Genome Sequence of Acinetobacter baumannii TYTH-1

Acinetobacter baumannii has emerged recently as a major cause of health care-associated infections due to the extent of its antimicrobial resistance and its propensity to cause large nosocomial outbreaks. Here we report the genome sequence of Acinetobacter baumannii TYTH-1 isolated in Taiwan during 2008.     

Discovery of potent antimicrobial peptide analogs of Ixosin-B

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 μM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.     

Loss of thioredoxin function in retinas of mice overexpressing amyloid β

Amyloid β peptides (Aβ) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aβ (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aβ-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.     

Effects of rosiglitazone on global ischemia-induced hippocampal injury and expression of mitochondrial uncoupling protein 2

We investigate the effect of rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma) with anti-inflammatory and anti-oxidative actions, on hippocampal injury and its roles in mitochondrial uncoupling protein 2 (UCP2) expression caused by transient global ischemia (TGI) in rats. Increased UCP2 expression was observed in mitochondria of hippocampal CA1 2-24h after TGI/reperfusion, with maximal expression levels at 6-18h. Administration of rosiglitazone to hippocampus 30min prior to the onset of TGI further enhanced mitochondrial UCP2 expression 2-6h following TGI/reperfusion. Rats subjected to TGI/reperfusion displayed a significant increase in lipid peroxidation, based on increased malondialdehyde (MDA) levels, in hippocampal CA1 mitochondria 2-6 h after reperfusion. Rosiglitazone significantly attenuated TGI/reperfusion-induced lipid peroxidation and suppressed hippocampal CA1 neuronal death based on the surviving neuronal counts. In conclusion, our results provide correlative evidence for the "PPARgamma-->UCP2-->neuroprotection" cascade in ischemic brain injury.     

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.     

Synthesis of zero-valent copper-chitosan nanocomposites and their application for treatment of hexavalent chromium

This study used ionotropic crosslinking to synthesize chitosan-tripolyphosphate chelating resin beads, which are used to fabricate zero-valent copper-chitosan nanocomposites. The copper nanoparticles were dispersed on chitosan-tripolyphosphate beads, and were thus able to maintain appropriate dispersion and stability, which greatly improves their applicability. The fabrication process contains two steps: using chitosan-tripolyphosphate beads to adsorb Cu(II) ions, followed by chemical reduction to reduce Cu(II) ions to zero-valent copper. This study explored the adsorption of synthesized chitosan-tripolyphosphate beads to Cu(II) ions, and used SEM/EDS, XPS, and TEM to examine the properties of zero-valent copper-chitosan nanocomposites. The results showed that, the adsorption behavior of hexavalent chromium from aqueous solution onto fabricated nanocomposites has better adsorption capacity than that of the chitosan-tripolyphosphate beads.