Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.
Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer’s disease and other tauopathies.
Estimation of apparent molecular weight (raw) of inactive renin by gel filtration of human plasma was found to be inaccurate when "acid activation" or "cryoactivation" was used for detection; recoveries were only 5% to 20%. Trypsln activation produced greater recoveries, but the apparent elution volume of inactive renin varied with the concentration of trypsln used; the presence of trypsln inhibitors increased trypsin requirements to 100 to 200 Mg/ml in the 60,000 dalton region, while low protein concentration in the 50,000 dalton region resulted in destruction of renin by as little as 10 Mg/ml trypsin. A composite trypsin-activated inactive renin peak corresponded to a raw of 56,000 ± 1500 daltons (104% to 120% recovery), while active plasma renin was 48,000 ± 2000 daltons. When this prorenln-like substance was isolated by affinity chromatography, it was found to be completely inactive. It was also nearly free of trypsin inhibitors, so that a single trypsin concentration correctly identified and confirmed the elution characteristics of inactive renin peak following gel filtration. The apparent mw of trypsin-activated inactive renin was slightly lower (52,500 daltons) than that of inactive renin.Human renal cortex was also found to contain a trypsin-actlvable form of renin. Like plasma inactive renin, it could be isolated by chromatography on Cibacron blue-agarose (Affi-Gel blue). It was found to be completely inactive following passage over a pepstatin affinity column. This inactive renal renin, as well as a similar substance in perfusate of normal human kidney, had a mw of 49,500 ± 1000, while active renal renin was 39,500 ± 500. Trypsin-activated inactive renal renin had a mw of 46,500 ± 500; its pH optimum was identical with that of active renal renin, and it no longer bound to Cibacron blue-agarose. We conclude that both human plasma and kidney contain an inactive, prorenin-like substance that can be detected reliably by trypsin activation. There appear to be slight differences in the apparent mw of plasma renins and kidney renin, but the similarity of other characteristics suggests that the inactive, prorenin-llke substances in renal cortex, renal perfusate, and plasma may be one and the same substance. (Hypertension 3 (suppl I): I-30-I-40, 1981) KEY WORDS • renin • prorenin • inactive renin • kidney • plasma affinity chromatography • molecular weight A N inactive form of renin (EC 3.4.99.19) inhuman plasma develops renin-like activity following limited proteolysis, including the processes of acid activation or cryoactivation. 13 As recently reviewed,' there has been considerable disagreement on the apparent molecular weight of inactive renin as determined by gel filtration of plasma, with values ranging from 43,000 to 63,000. 4 -10 These discrepancies may be related in part to the use of acid activation to detect inactive renin in chromatographic fractions, since the activation of inactive renin that occurs at pH 3 is reversible at a higher pH 11 ' 12 and the subsequent activation that occurs at alkaline ...
Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.
As bstract. Inactive renin comprises well over half the total renin in normal human plasma. There is a direct relationship between active and inactive renin levels in normal and hypertensive populations, but the proportion ofinactive renin varies inversely with the active renin level; as much as 98% of plasma renin is inactive in patients with low renin, whereas the proportion is consistently lower (usually 20-60%) in high-renin states. Two hypertensive patients with proven renin-secreting carcinomas of non-renal origin (pancreas and ovary) had high plasma active renin (1 19 and 138 ng/h per ml) and the highest inactive renin levels we have ever observed (5,200 and 14,300 ng/h per ml; normal range 3-50). The proportion ofinactive renin (98-99%) renins isolated from plasma and tumors ofthese patients were biochemically similar to semipurified inactive renins from normal plasma or cadaver kidney. All were bound by Cibacron Blue-agarose, were not retained by pepstatinSepharose, and had greater apparent molecular weights (Mr) than the corresponding active forms. Plasma and tumor inactive renins from the three patients were similar in size (Mr 52,000-54,000), whereas normal plasma inactive renin had a slightly larger Mr than that from kidney (56,000 vs. 50,000). Inactive renin from each source was activated irreversibly by trypsin and reversibly by dialysis to pH 3.3 at 40C; the reversal process followed the kinetics of a first-order reaction in each instance. The trypsinactivated inactive renins were all identical to semipurified active renal renin in terms of pH optimum (pH 5.5-6.0) and kinetics with homologous angiotensinogen (Michaelis constants, 0.8-1.3 AM) and inhibition by pepstatin or by serial dilutions of renin-specific antibody. These results indicate that a markedly elevated plasma inactive renin level distinguishes patients with ectopic renin production from other high-renin hypertensive states. The co-production of inactive and active renin by extrarenal neoplasms provides strong presumptive evidence that inactive renin is a biosynthetic precursor of active renin. The unusually high proportion of inactive renin in plasma and tumor extracts from such patients is consistent with ineffective precursor processing by neoplastic tissue, suggesting that if activation of "prorenin" is involved in the normal regulation of active renin levels it more likely 437 Prorenin in Nonrenal Neoplasms, Plasma, and Kidney J. Clin. Invest.
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