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.

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 Nα-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.

Hypoxia-inducible factor 1alpha- mediated resistance to phenolic anticancer.

BACKGROUND: Phenolic compounds EGCG [(-)-epigallocatechin-3-gallate], resveratrol (3,4',5-trihydroxy-trans-stilbene) and capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) are worth investigating for clinical application in cancer prevention and chemotherapy. Hypoxia-induced drug resistance is a major obstacle in the development of effective cancer chemotherapy. Therefore, we examined whether drug resistance to these phenolic compounds is acquired by hypoxia. METHODS: Hep3B hepatoma, Caki-1 renal carcinoma, SK-N-MC neuroblastoma, and HEK293 cell lines were cultured under normoxic or hypoxic conditions. Drug sensitivities to the phenolic compounds and expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and the multidrug resistance genes were examined in these cell lines. RESULTS: Drug resistance was acquired 24 h after hypoxia and subsided 8 h after reoxygenation. Protein synthesis inhibitors abolished this drug resistance. A transfection study demonstrated that HIF-1alpha enhanced this hypoxia-induced resistance and that its dominant-negative isoform suppressed resistance acquisition. However, MDR1 and MRP1, which provide multidrug resistance to conventional anticancer agents, were not induced by hypoxia. CONCLUSIONS: These results suggest that HIF-1alpha-dependent gene expression participates in the cellular process of the hypoxia-induced resistance to phenolic compounds.

Hypoxic activation of the atrial natriuretic peptide gene promoter through direct and indirect actions of hypoxia-inducible factor-1.

Atrial natriuretic peptide (ANP) is a cardiac peptide, the transcription of which is up-regulated in the ischaemic ventricle. However, the molecular mechanism of ANP induction is unclear. This study demonstrated that ANP mRNA expression in rat ventricular myocardium is induced in an early phase of ischaemia, preceded by hypoxia-inducible factor-1 (HIF-1) alpha expression. The ANP gene was also induced by hypoxia or HIF-1 inducers such as CoCl2 and desferrioxamine in H9c2 and neonatal cardiomyocytes. The 2307 bp 5'-flanking region of the rat ANP gene was cloned and fused to the luciferase gene. Evidence of the promoter activity was only apparent in the myocytes and was induced by hypoxia and HIF-1 inducers. The overexpression of HIF-1alpha markedly enhanced ANP promoter activity, and a dominant-negative isoform completely suppressed it. We demonstrated that the promoter regions are essential for hypoxic ANP induction. One promoter region, containing the HIF-1-binding sequence, is regulated directly by HIF-1. The other region is also activated by HIF-1 despite having no HIF-1-binding sequence. These results suggest that HIF-1 enhances the transactivation of the ANP gene in hypoxic myocytes, implying that stimulation of the ANP promoter by HIF-1 may in fact be responsible for the induction of the ANP gene in ischaemic ventricular myocardium.