Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

A general method was developed for the discovery of protease‐activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease‐2 substrate linker to a vascular endothelial growth factor‐binding peptide and sorted using a two‐stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease‐mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100‐fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.     

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 μM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.     

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adiacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH₂F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.     

Effect of different collagenases on the isolation of viable hepatocytes from rat liver

The isolation of viable hepatocytes from rat liver was found to depend on the source of collagenase (EC 3.4.24.3) more than any other single factor examined. Collagenase purified from Achromobacter iophagus/Bacillus polymyxa (collagenase/dispase) gave reproducibly high viability without the use of complex perfusion protocols.     

Serum Deprivation and Protein Synthesis Inhibition Induce Two Different Apoptotic Processes in N18 Neuroblastoma Cells

N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.     

Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor inLeishmania donovani

A calcium dependent proteolytic enzyme was detected in the lysed promastigotes ofLeishmania donovani, the causative agent of kala-azar. The protease was able to hydrolyse and added substrate, azocasein and showed high affinity for calcium. Rate of azocasein digestion was primarily slow but boosted up after eight hours. It was not inactivated when heated at 55° C for 15 min at pH 7.4. Sulfhydryl reagents significantly reduced the enzymic activity but trypsin-like protease inhibitors hardly had any effect. The enzyme was not sensitive to calmodulin from a heterologous source but registered low activity when treated with chlorpromazine. The caseinolytic activity was stimulated when leishmanial cells were preincubated with ionophore A23187 in presence of 1 mM Ca²⁺. The enzyme is named caldonopain due to its similarity with a general class of calcium dependent protease calpain present in different tissues and cells. Caldonopain was found to be localized in cytosol along with its specific endogenous inhibitor caldonostatin. The ratio of caldonopain-caldonostatin unit was higher in the infected macrophage compared to the parasitic protozoa and Balb/c macrophage alone. It may be postulated that the amount of both calcium and its protein inhibitor may have a direct impact on the caldonopain-induced biological process to regulate cellular action of this pathogen.Part of this work was presented at the International Symposium on Current Trends in Leishmania Research held in February, 1992 at Calcutta, India     

Synthesis of Ethanolamine Phosphoglycerides in Rat Cerebral Cortex Subjected In Vitro to Experimental Hypoxia with and without Hypocapnia

Slices and homogenates from rat cerebral cortex were used to study the effect of hypoxia, with or without hypocapnia, on phosphatidylethanolamine synthesis. The incorporation of [1-³H]ethanolamine into the corresponding phospholipid was greatest in slices treated with pure nitrogen, intermediate when the nitrogen contained 5% CO₂, and least in slices treated with 95% O₂-5% CO₂. The role of hypocapnia in reinforcing the effect due to hypoxia did not require the integrity of the cell because similar results were obtained by treating homogenates with pure nitrogen or nitrogen plus 5% CO₂. In both cases the synthesis of phosphatidylethanolamine was abolished by the addition of EGTA and the degradation of newly synthesized phospholipid by phospholipases was similar to that obtained in controls. When the homogenate was not buffered, changes in the pH due to experimental treatment influenced the response to Ca²⁺ and to hypoxia plus hypocapnia. Intracellular calcium ions are thought to play a role in the response of cerebrocortical slices to N₂-treatment. In fact, although the incorporation was greater in complete medium that contains 2 mM Ca²⁺ than in the same medium prepared without the addition of this ion, the relative increase of incorporation due to N₂-treatment was greater in the medium lacking added Ca²⁺.     

Solvent accessibility as a predictive tool for the free energy of inhibitor binding to the HIV-1 protease.

We have developed a simple approach for the evaluation of the free energies of inhibitor binding to the protease of the human immunodeficiency virus (HIV-1 PR). Our algorithm is based on the observation that most groups that line the binding pockets of this enzyme are hydrophobic in nature. Based on this fact, we have likened the binding of an inhibitor to this enzyme to its transfer from water to a medium of lower polarity. The resulting expression produced values for the free energy of binding of inhibitors to the HIV-1 PR that are in good agreement with experimental values. The additive nature of this approach has enabled us to partition the free energy of binding into the contributions of single fragments. The resulting analysis clearly indicates the existence of a ranking in the participation of the enzyme's subsites in binding. Although all the enzyme's pockets contribute to binding, the ones that bind the P2-P'2 span of the inhibitor are in general the most critical for high inhibitor potency. Moreover, our method has allowed us to determine the nature of the functional groups that fit into given enzyme binding pockets. Perusal of the energy contributions of single side chains has shown that a large number of hydrophobic and aromatic groups located in the central portion of the HIV-1 PR inhibitors present optimal binding. All of these observations are in agreement with experimental evidence, providing a validation for the physical relevancy of our model.     

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.