A bridge between the RNA and protein worlds? Accelerating delivery of chemical reactivity to RNA and DNA by a specific short peptide (AAKK)(4).

BACKGROUND: RNA can catalyze diverse chemical reactions, leading to the hypothesis that an RNA world existed early in evolution. Today, however, catalysis by naturally occurring RNAs is rare and most chemical transformations within cells require proteins. This has led to interest in the design of small peptides capable of catalyzing chemical transformations. RESULTS: We demonstrate that a short lysine-rich peptide (AAKK)(4) can deliver a nucleophile to DNA or RNA and amplify the rate of chemical modification by up to 3400-fold. We also tested similar peptides that contain ornithine or arginine in place of lysine, peptides with altered stereochemistry or orientation, and peptides containing eight lysines but with different spacing. Surprisingly, these similar peptides function much less well, suggesting that specific combinations of amino acids, charge distribution, and stereochemistry are necessary for the rate enhancement by (AAKK)(4). CONCLUSIONS: By appending other reactive groups to (AAKK)(4) it should be possible to greatly expand the potential for small peptides to directly catalyze modification of DNA or RNA or to act as cofactors to promote ribozyme catalysis.

Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates.

BACKGROUND: Understanding the networks of selective proteolysis that regulate complex biological systems requires an appreciation of the molecular mechanisms used to maintain substrate specificity. Human plasmin, a serine protease that promotes the dissolution of blood clots and is essential in maintaining normal hemostasis, is usually described as having broad substrate specificity. Recent evidence that plasmin also plays a key role in a variety of other important biological and pathological processes, however, has suggested that this description might need to be re-evaluated. RESULTS: We used substrate phage display to elucidate optimal subsite occupancy for substrates of plasmin. We identified a peptide substrate that is cleaved 710,000-fold more efficiently by plasmin than a peptide containing the activation sequence of plasminogen. Plasmin achieves this unexpected, large differential activity even though both target sequences possess an arginine residue in the P1 position. We also demonstrate that proteolysis by plasmin can be targeted to an engineered protein substrate and that introduction of substrate sequences identified by phage display into plasminogen increases plasmin-mediated cleavage of the mutant 2000-fold. CONCLUSIONS: The specificity of plasmin is more tightly controlled than previously recognized; interactions with substrates at all subsites between S4 and S2' contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This 'negative selectivity' avoids short-circuiting regulation of the fibrinolytic system and other important biological processes, and might be an important general mechanism for controlling protease cascades.

Substrate specificity of prostate-specific antigen (PSA).

BACKGROUND: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage. RESULTS: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. CONCLUSION: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.

Numbers of gastrointestinal helminth eggs of Wyoming cattle in two surveys: 1957 to 1961 and 1973 to 1977.

From 1973 to 1977, a survey of internal parasites in Wyoming cattle was conducted via fecal analyses of 1,490 beef cattle. Sugar flotation techniques were used, with a factor of 2 times the actual egg counts. The prevalence of internal parasites of beef cattle in this survey was compared with that in a previous survey conducted on Wyoming beef cattle from 1957 to 1961. Results of analyses indicated no true change in mean eggs per gram of feces (epg) as follows: calves, 14 epg in 1961 and 20 epg in 1977; yearlings, 29 epg in 1961 and 19 epg in 1977; and adults, 22 epg in 1961 and 21 epg in 1977.

Coccidiostatic action of monensin fed to lambs: body weight gains and feed conversion efficacy.

Thirty crossbred ewe lambs weighing an average of 37.3 kg were allotted to 6 groups of 5 lambs each so that group weights were nearly equal. Lambs were fed dehydrated alfalfa pellets, initially at 1.14 kg/day and subsequently increased after experimental day 15 and 42. Each lamb was artificially infected with 18,000 sporulated oocysts of Eimeria ninakohlyakimovae. Groups 1, 2, 3, and 4 were given monensin in the form of medicated alfalfa pellets at dose levels of 5, 10, 20, and 30 g/metric ton, respectively. Groups 5 and 6 were infected controls (infected, nonmedicated). Lambs in groups 5 and 6 developed severe clinical coccidiosis, having diarrhea and losing weight rapidly. Group 1 lambs did not have diarrhea, but the lambs did not gain well. All other medicated lambs gained weight during the experimental period of 84 days. Feed conversion was good in medicated groups 2, 3, and 4 and was poor in control groups 5 and 6. Statistically significant differences in feed conversion and body weight gains (5 and 1% level of confidence) were observed between control and medicated groups.

Parasitism (Trichostrongylus colubriformis and Eimeria ninakohlyakimovae) in sheep: relationship between wool fiber diameter changes and feed conversion efficiency.

Twenty-five lambs, 5 months of age, were used to compare the infection pressure of Trichostrongylus colubriformis with that of Eimeria ninakohlyakimovae. Effects of the parasitic infection pressure were assayed by determining body weight gains, feed conversion efficiencies, and wool fiber diameter changes (reflecting changes in protein metabolism) in lambs fed a good ration and those given a marginal diet (dehydrated alfalfa pellets). Significant differences in weight grains or feed conversion efficiencies of nematode- or coccidia-infected lambs and noninfected controls were not found, but significant differences due to the parasites' effect on wool fiber diameters and in lamb response to different feeds were found. Protein uptake or assimilation, or both, were apparently affected by a combination of infection with E ninakohlyakimovae and a marginal diet.

Diagnosis of Corynebacterium pseudotuberculosis infections in sheep, using an enzyme-linked immunosorbent assay.

Lambs infected with Corynebacterium pseudotuberculosis (ovis) by injecting suspensions of live bacteria into the wool-free area of the axilla developed antibody against cell wall antigens and antitoxin, as measured by the enzyme-linked immunosorbent assay. The toxin was a better antigen for measuring infection than was the cell wall antigen. The enzyme-linked immunosorbent assay appeared to be as sensitive as the antihemolysin inhibition test for detecting antitoxin and was easier to perform.

Prevalence of Dictyocaulus viviparus infection in Rocky Mountain elk in Teton County, Wyoming.

Dictyocaulus viviparus infections in Rocky Mountain elk (Cervus canadensis of Teton Countywere surveyed by fecal analyses during spring, summer and winter and by fecal analyses and necropsies during fall hunting seasons, 1968-1973. Prevalance of the lungworms was relatively high: 32-70% during the spring; slightly lower, 30-47%, during the summer; 21-39% in the fall; and declined to the annual low of 8-19% during the winter. Conversely, elk summering on Big Game Ridge showed an increase in prevalance of D. viviparus from 1969 to 1973. Decreases in prevalance of lungworms were noted on the National Elk Refuge at Jackson after management changes were effected in 1971.

Bovine coccidia in American bison.

Three species of coccidia, found in American bison sampled in Wyoming, are identified. The described coccidial species, common to cattle, have not been reported previosuly from American bison, (Bison bison). Identification of the parasites was determined by oocyst structural measurements and by oocyst sporulation times.

Directing sequence-specific proteolysis to new targets. The influence of loop size and target sequence on selective proteolysis by tissue-type plasminogen activator and urokinase-type plasminogen activator.

We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). We demonstrate that this information can be used to direct selective proteolysis to new protein targets. Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Omega) loop of staphylococcal nuclease. Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and Km values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility. Cleavage of proteins by t-PA and u-PA is sequence selective. u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.

The structure of a Michaelis serpin-protease complex.

Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.