A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates.

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata.

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport.

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from approximately 3min in L. maderae to approximately 25min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes.

Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4-8 region.

Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr(5), and the importance of positions 4 and 8. For example, when Trp(8) and Phe(4) were exchanged, the resulting analogue (Trp(4),Phe(8)-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe(4), with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp(8) (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp(8) (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.

Synthesis and biological activity of adipokinetic hormone analogues modified at the C-terminus.

A series of Locusta adipokinetic hormone I (AKH-I), < QLNFTPNWGTa, analogues, were synthesized with modifications at the C-terminal threonine residue using a combination of solid- and liquid-phase methodology and evaluated in Locusta migratoria, in a lipid mobilization assay in vivo and an acetate uptake assay in vitro. Modifications at Thr10 of AKH-I involved replacement of its C-terminal amide by the groups -OH, -OCH3, -NHCH3, -N(CH3)2, and -NHC6H5; the last three groups were also applied to the amide of AKH-I-[Thr(Bzl)10]. The methyl ester, monomethyl, and dimethyl analogues were all of lower activity than the parent in the lipid mobilization assay, but lost less than two orders of potency. In the acetate uptake assay, again the methyl ester analogue showed the greatest retention of biological activity of all modified peptides. A cyclic analogue, cyclo (PLNFTPNWGT), was active in both assays, but only at very high concentrations. Almost all analogues were more active in the acetate uptake assay than in the lipid assay, but unusually, AKH-I-NHCH, and AKH-I-N(CH3)2, together with cyclo(PLNFTPNWGT), were more active in the lipid mobilization assay. In addition, the acid AKH-I analogue did not suffer as large a loss in potency in the lipid mobilization assay as in the acetate uptake assay, although it was less potent in the former. The relative potencies of these two methyl analogues contrast with those for AKH-I[Thr(Bzl)10]-NHCH3 and AKH-I-[Thr(Bzl)10]-N(CH3)2, which, together with both phenyl analogues, were significantly more active in the acetate uptake assay. We conclude that the acetate uptake assay has a greater preference for a hydrophobic C-terminus, compared with the lipid mobilization assay.