A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome.

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a key regulator of cell-cycle progression and its expression is tightly regulated at the level of transcription and by proteasome-dependent proteolysis. The turnover of p21WAF1/CIP1 by proteasomes does not always require the ubiquitylation of p21WAF1/CIP1 suggesting that there could be an alternative pathway into the proteasome. Here we show that the C8 alpha-subunit of the 20S proteasome interacts with the C-terminus of p21WAF1/CIP1 and mediates the degradation of p21WAF1/CIP1. A small deletion in this region that disrupts binding to C8 increased the half-life of p21WAF1/CIP1 expressed in vivo. In contrast a deletion that increased the affinity between C8 and p21WAF1/CIP1 significantly reduced the stability of the latter. These data suggest that interaction with a 20S proteasome alpha-subunit is a critical determinant of p21WAF1/CIP1 turn-over and show how non-ubiquitylated molecules might bypass the 19S regulator of the proteasome and become targeted directly to the 20S, core protease. Consistent with this, p21WAF1/CIP1 was degraded rapidly by purified 20S proteasomes in a manner that was dependent on the C8-interaction domain.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

New nonpeptide angiotensin II receptor antagonists. 3. Synthesis, biological properties, and structure-activity relationships of 2-alkyl-4-(biphenylylmethoxy)pyridine derivatives.

A novel series of nonpeptide angiotensin II (AII) receptor antagonists is reported, derived from linkage of the biphenylyltetrazole moiety found in previously described antagonists via a methyleneoxy chain to the 4-position of a 3-substituted 2,6-dialkylpyridine. When evaluated in an in vitro binding assay using a guinea pig adrenal membrane preparation, compounds in this series generally gave IC50 values in the range 0.005-0.5 microM. A variety of substituents was found to be effective at the 3-position of the pyridine ring. On intravenous administration in a normotensive rat model, the more potent compounds inhibited the AII-induced pressor response with ED50 values in the range 0.1-1.0 mg/kg. One of the compounds, 2-ethyl-5,6,7,8-tetrahydro-4-([2'-(1H-tetrazol-5-yl)biphenyl-4y l] methoxy)quinoline (26), demonstrated good oral activity in two rat models. At doses in the range 1-10 mg/kg po in AII-infused, conscious, normotensive rats, the compound exhibited a dose-related inhibition of the pressor response with a good duration of action at the higher doses. In a renal hypertensive rat model compound 26 showed a rapid and sustained lowering of blood pressure at a dose of 5 mg/kg po. Based on its profile, this compound, designated ICI D6888, has been selected for evaluation in volunteers.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 3.1 Synthesis and biological properties of aminodeoxystatine and difluorostatone derivatives.

Two series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity have been synthesized which incorporate the transition-state mimetics (3S,4S)- and (3R,4S)-5-cyclohexyl-3,4-diaminopentanoic acid ((S)- and (R)-CDAPA), and (4S)-4-amino-5-cyclohexyl-2,2-difluoro-3-oxopentanoic acid (ACDFOPA). Several compounds in these series, for example 13a, 19c, and 19f, were highly potent inhibitors of partially purified human renin (IC50 values of 3.9, 1.6, and 1.4 nM, respectively). The ACDFOPA-based compounds 19c and 19f contain no natural amino acid fragments and have molecular weights which compare well with those of previously reported inhibitors of nanomolar in vitro potency. When administered intravenously to anesthetized, sodium-depleted marmosets at doses of 3 mg/kg, compounds 13a and 19c caused a marked reduction in mean arterial pressure, but in the same animal model at 30 mg/kg, oral activity was not seen.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 2. Synthesis, biological properties and molecular modeling of hydroxyethylene isostere derivatives.

A series of inhibitors of human renin have been synthesized, derived from combination of a 2-(8-propyl-6-pyridin-3-yl-1,2,4-triazolo[4,3-a]pyrazin-3-yl)- 3-pyridin- 3-ylpropionic acid moiety 6c with the hydroxyethylene isostere of the scissile amide bond (2S,4S,5S)-5-amino-6-cyclohexyl-4-hydroxy-2-isopropylhexanoic acid (ChaOH--Val). The more potent members of this series showed good inhibitory activity against partially purified human renin, 7d, for example, having an IC50 of 0.2 nM. Structure-activity relationships for these compounds were consistent with their binding to the S4-S2' sites of human renin. Analogues 7e and 7h-k with a variety of substituents at the C-terminus all had in vitro IC50S less than 1 nM. In contrast with the majority of previously reported inhibitors of similar potency, these compounds contain no natural amino acid fragments. When administered intravenously to anesthetized, sodium-depleted marmosets at doses of 0.3-3.0 mg/kg, compound 7d caused a marked reduction in mean arterial pressure. Following oral administration at 30 mg/kg in the same animal model, 7d again elicited a significant fall in mean arterial pressure, accompanied by suppression of plasma renin activity lasting up to 3 h after dosing.

Aberrant coronary artery in association with a quadricuspid pulmonary valve.

A case of quadricuspid pulmonary valve with an accessory coronary artery in an 82 year old woman is reported. This represents persistance of an early embryonic stage of development of the coronary circulation.

Static mechanical properties of the developing and mature rat aorta.

The static mechanical properties of the aorta have been examined in rats, aged between 4 weeks and 2 years. Internal radius, relative wall thickness, circumferential incremental strain, and circumferential incremental elastic modulus have been measured at pressures between 1.33 and 33.3 kPa (10 and 250 mm Hg). The results show that the value of the incremental elastic modulus does not change after 12 weeks. Changes observed in younger animals are related to alterations in relative wall thickness. The findings correlate with previously observed chemical and morphological changes. Studies of muscle function indicate that the smooth muscle of the media does not contribute significantly to the static elastic properties of the vessel wall.