Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA N^α-terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway

Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate’s degradation. We discovered that the human Nt-Asn–specific Nt-amidase NTAN1, Nt-Gln–specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate’s dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron–targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed “superchanneling.” In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate’s sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex

Polyelectrolyte complex micelles by self-assembly of polypeptide-based triblock copolymer for doxorubicin delivery

AbstractPolyelectrolyte complex micelles were prepared by self-assembly of polypeptide-based triblock copolymer as a new drug carrier for cancer chemotherapy. The triblock copolymer, poly(l-aspartic acid)-b-poly(ethylene glycol)-b-poly(l-aspartic acid) (PLD-b-PEG-b-PLD), spontaneously self-assembled with doxorubicin (DOX) via electrostatic interactions to form spherical micelles with a particle size of 60–80 nm (triblock ionomer complexes micelles, TBIC micelles). These micelles exhibited a high loading capacity of 70% (w/w) at a drug/polymer ratio of 0.5 at pH 7.0. They showed pH-responsive release patterns, with higher release at acidic pH than at physiological pH. Furthermore, DOX-loaded TBIC micelles exerted less cytotoxicity than free DOX in the A-549 human lung cancer cell line. Confocal microscopy in A-549 cells indicated that DOX-loaded TBIC micelles were transported into lysosomes via endocytosis. These micelles possessed favorable pharmacokinetic characteristics and showed sustained DOX release in rats. Overall, these findings indicate that PLD-b-PEG-b-PLD polypeptide micelles are a promising approach for anti-cancer drug delivery

Effect of rhBMP-2 applied with a 3D-printed titanium implant on new bone formation in rabbit calvarium

Objective: This study sought to compare the biocompatibility of a three-dimensional (3D)-printed titanium implant with a conventional machined titanium product, as well as the effect of such implant applied with recombinant human Bone Morphogenetic Protein Type 2 (rhBMP-2) for guided bone regeneration. Methodology: Disk-shaped titanium specimens fabricated either by the conventional machining technique or by the 3D-printing technique were compared by MC3T3-E1 cells cytotoxicity assay. New bone formation was evaluated using a rapid prototype titanium cap applied to the calvaria of 10 rabbits, which were divided into two groups: one including an atelopeptide collagen plug on one side of the cap (group I) and the other including a plug with rhBMP-2 on the other side (group II). At six and 12 weeks after euthanasia, rabbits calvaria underwent morphometric analysis through radiological and histological examination. Results: Through the cytotoxicity assay, we identified a significantly higher number of MC3T3-E1 cells in the 3D-printed specimen when compared to the machined specimen after 48 hours of culture. Moreover, morphometric analysis indicated significantly greater bone formation at week 12 on the side where rhBMP-2 was applied when evaluating the upper portion immediately below the ca p. Conclusion: The results suggest that 3D-printed titanium implant applied with rhBMP-2 enables new bone formation

Electrical current suppression in Pd-doped vanadium pentoxide nanowires caused by reduction in PdO due to hydrogen exposure

Pd nanoparticle-doped vanadium pentoxide nanowires (Pd-VONs) were synthesized. Electrical current suppression was observed when the Pd-VON was exposed to hydrogen gas, which cannot be explained by the work function changes mentioned in previous report such as Pd-doped carbon nanotubes and SnO 2 nanowires. Using the x-ray photoelectron spectroscopy, we found that the reduction in PdO due to hydrogen exposure plays an important role in the current suppression of the Pd-VON.open4

Explaining the continuum of social participation among older adults in Singapore: from 'closed doors' to active ageing in multi-ethnic community settings.

OBJECTIVES: This study aims to identify and explain the continuum in which older people in Singapore participate in community and social life, highlighting the influence of culture and policy context on social participation. METHODS: Using an ethnographic approach in a neighbourhood (n=109), we conducted focus groups with older adults of different ethnicities, exploring experiences of social participation. Next, participants took 50 photographs relating to 'lives of elders', showcasing the socio-ecological context that influenced social participation. Lastly, go-along interviews were conducted in various precincts with community leaders. RESULTS: A continuum of social participation emerged among older adults, ranging from (1) marginalization and exclusion, to (2) 'comfort-zoning' alone (3) seeking consistent social interactions, (4) expansion of social network, and (5) giving back to society. Seeking consistent social interactions was shaped by a preference for cultural grouping and ethnic values, but also a desire for emotional safety. Attitudes about expanding one's social network depended on the psychosocial adjustment of the older person to the prospect of gossip and 'trouble' of managing social relations. Despite the societal desirability of an active ageing lifestyle, cultural scripts emphasizing family meant older adults organized participation in social and community life, around family responsibilities. Institutionalizing family reliance in Singapore's welfare approach penalized lower-income older adults with little family support from accessing subsidies, and left some living on the margins. DISCUSSION: To promote inclusiveness, ageing programs should address preferences for social participation, overcoming barriers at the individual, ethnic culture and policy level

Activation of Protein Kinase G After Repeated Cocaine Administration Is Necessary for the Phosphorylation of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor GluA1 at Serine 831 in the Rat Nucleus Accumbens

Phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the striatum plays a crucial role in regulating the receptor-coupled signaling cascades leading to behavioral changes associated with psychostimulant exposure. The present study determined if activation of protein kinase G (PKG) contributes to the phosphorylation of AMPA receptor GluA1 subunit at the position of serine 831 (GluA1-S831) in the rat nucleus accumbens (NAc) after repeated cocaine administration. The results demonstrated that repeated intraperitoneal (i.p.) injections of cocaine (20 mg/kg) once a day for seven consecutive days significantly increased the level of phosphorylated (p)GluA1-S831. This increase was decreased by the intra-NAc infusion of either the cyclic guanosine monophosphate (cGMP) analog, Rp-8-Br-PET-cGMPS (5 nmol/1 μL), or the PKG inhibitor, KT5823 (2 nmol/1 μL). Repeated cocaine administration increased PKG binding activity to GluA1. This increase in GluA1-S831 phosphorylation after repeated cocaine administration was decreased by the intra-NAc infusion of the synthetic peptide (Tat-tagged interfering peptide (Tat-GluA1-i)), that interferes with the binding of PKG to GluA1. Intra-NAc infusion of the interfering peptide also reduced the repeated cocaine-induced increase in locomotor activity. These findings suggest that activated PKG, after repeated exposure to cocaine, binds to AMPA receptor GluA1 and is required for the phosphorylation of S831, contributing to behavioral changes