Changes In Peptidase Activities Research Articles

Characterization of a Kunitz trypsin inhibitor from Enterolobium timbouva with activity against Candida species

Scientific advances have not been sufficient to accompany the growing resistance to antimicrobial medicines. High mortality rates due to opportunistic infections have threated human health. The development of new drugs, such as those obtained from plant sources, is a world priority. Herein, we report the purification of a trypsin inhibitor from Enterolobium timbouva seeds (EtTI) with regard to its homology, physico-chemical and inhibitory properties. Furthermore, we evaluated its activity against Candida strains, opportunistic pathogens regularly found in hospital infections. EtTI belongs to the Kunitz family and inhibits two trypsin molecules simultaneously; a feature shared among double-headed Kunitz inhibitors. A high inhibitory activity against trypsin was determined (Ki = 0.5 nM), and refractory to digestion by pepsin. EtTI was candidicidal against C. albicans, C. buinensis and C. tropicalis, triggering disturbances on integrity of the plasma membrane and morphological alterations, presumably mediated via apoptosis. The presence of two reactive sites is an unusual feature detected in EtTI. Numerous diseases and pathologies involve changes in peptidase activities, encouraging studies with multifunctional inhibitors. Accordingly, the further exploration of EtTI could provide new insights into the Kunitz inhibitors and their applications in disease control.

Seasonal changes in peptidase activities and their properties in the surface water of Lake Shinryu

Serial monthly peptidase activities were assayed in the surface water of Lake Shinryu, located in the Chugoku district of Japan, using artificial fluorescent peptidase substrates. The results indicated that the lake water had higher aminopeptidase activities than endopeptidase activities except in November and December, whereas lake water filtrated using membranes with a pore size of 0.2 μm showed higher endopeptidase activities than aminopeptidase activities from June to December, with peaks in June and November. The serial aminopeptidase activity profile was relatively similar to that of the chlorophyll-a concentration. The size distribution of aminopeptidase activities indicated that half of the total activity was retained in the fraction between 5 and 100 μm. These results suggest that phytoplankton participate in the digestion of peptides with aminopeptidase activities. Electrophoretic analysis detected the presence of three peptidase bands (1, 2, and 3) in the lake water that passed through the 0.2-μm membranes. Samples from June, September, and November dominantly contained these active bands in different electrophoretic profiles.

Impact of ageing on proteasome structure and function in human lymphocytes

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major “housekeeping” proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20–63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Recent studies have implicated proteasomes in the generation of the antigenic peptides that are presented on major histocompatibility complex class I molecules to T lymphocytes. Interferon gamma modifies the subunit composition of proteasomes and causes changes in their peptidase activities that should favor the production of peptides with hydrophobic or basic carboxyl termini (i.e., the types found on major histocompatibility complex class I molecules). It has been proposed that these changes in peptidase activity are due to incorporation into proteasomes of the major histocompatibility complex-encoded subunits LMP2 and -7, which are induced by interferon gamma. Here we show by gene transfection into lymphoblasts or HeLa cells that LMP7 increases the capacity (Vmax) of 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues without affecting hydrolysis after acidic residues. These changes depended on the amount of LMP7 subunits incorporated into proteasomes. Transfection of LMP2 reduced cleavage of peptides after acidic residues, increased hydrolysis after basic residues, and did not affect the hydrophobic activity. Since the activity of the total proteasome population changed after incorporation of only small amounts of LMP2 or -7, these subunits must cause major alterations in peptidase activity. Thus, their expression can account for the changes in proteasome activity induced by inteferon gamma, and these findings lend further support to the proposed roles of LMPs in altering the nature of the peptides generated for antigen presentation.

Role of Proteasomes in Antigen Presentation

Recent studies have demonstrated that the proteasome, in addition to functioning in the complete degradation of cell proteins, is the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8- to 9-amino acid fragments, which are then transported into the endoplasmic reticulum, where they become associated with MHC-class I molecules and are thus delivered to the cell surface. A variety of evidence has shown that the proteasome and ATP-ubiquitin-dependent pathway are critical in this process: (1) In cells, selective inhibitors of proteasome function inhibit the bulk of protein degradation and thus prevent the generation of peptides necessary for class I presentation and the appearance of MHC on the cell surface. (2) Mutations that block ubiquitin conjugation prevent the generation of an antigenic peptide. (3) Modifications that lead to rapid degradation of a protein by the ubiquitin pathway enhance antigen presentation. (4) gamma-Interferon (gamma-IFN) induces new proteasome subunits, LMP2 and LMP7, encoded in the MHC region that are incorporated in place of constitutive proteasome subunits. Their incorporation does not affect rates of protein breakdown but causes changes in peptidase activities, i.e. they increase rates of cleavage after basic and hydrophobic residues and decrease cleavage after acidic residues. Transfections of cells with LMP2 or LMP7 cause similar changes in these peptidase activities as are caused by gamma-IFN. These modifications in peptidase activities should enhance the production of those types of peptides which are preferentially transported into endoplasmic reticulum and selectively bound to MHC-class I molecules.

Genetic and environmental effects on the expression of peptidases and larval viability in Drosophila melanogaster.

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.

Peptidases Are Affected Differently in Neocortical Regions of Brains from Patients with Alzheimer's Disease

Although it is recognized that changes in protease activities may be involved in the etiology of pathological changes in the human brain, there have been few studies on normal and pathological protein catabolism. In the present study the activities of major cortical aminopeptidase and prolylendopeptidase were determined in frontal and medial temporal cortical samples of brains from patients with Alzheimer''s disease (AD) and control patients matched for age. Prolylendopeptidase activity was reduced by 45–50% in the frontal and medial temporal cortex of AD patients as compared with controls, whereas major aminopeptidase activity was unaltered. The possibility that the changes in peptidase activities in AD are age-dependent was studied in postmortem neocortical samples from 18 AD patients who had died between 55 and 90 years of age. Prolylendopeptidase activity was lower in patients who died at a relatively early age than in patients who died at a relatively old age. This underscores the notion that, with respect to age, there may be heterogeneity in neurochemical changes in AD.

Circadian variations of vasopressin level and vasopressin-converting aminopeptidase activity in the rat pineal gland

Vasopressin levels and vasopressin-converting aminopeptidase activity were measured in the rat pineal gland during the 24 hr light-dark cycle. A rhythmic variation in peptide levels and peptidase activity occurred. At the onset of light at 6.00 hr, the peptidase displayed a significant, short-lasting (approximately 3 hr) increase of about 35% in activity, while a decrease of 28% in pineal vasopressin levels was observed. The changes in peptidase activity and peptide level were not triggered by light per se, since they persisted to occur at the same time point in animals which were not exposed to light, indicating the circadian nature of the rhythmicity. These changes were specific to the pineal gland, since other tissues, like hippocampus and pituitary gland, did not show these daily variations. The data suggest a relationship between vasopressin levels and vasopressin-converting aminopeptidase activity.

Peptidase activities in the functioning jejunum and ileum before and after jejunoileal bypass in morbid obesity.

Different peptidase activities were studied preoperatively and postoperatively on intestinal biopsy specimens from patients with jejunoileal bypass for morbid obesity. Preoperatively the activities of the brush border peptidases, microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.X), were lowest at the ligament of Treitz and highest in the distal ileum. The activity of the other brush border peptidase studied, the gamma-glutamyl transpeptidase (EC 2.3.2.2), had a maximum at proximal jejunum, like the two cytosol dipeptidases glycyl-leucine dipeptidase (EC 3.4.13.2) and proline dipeptidase (EC 3.4.13.9). Postoperative changes in peptidase activities were most pronounced in the ileal part of the shunt, whereas changes were small in the jejunal part. The most conspicuous finding was a significant increase in gamma-glutamyl transpeptidase activity in the ileum. There was also tendency to an increase of the cytosol dipeptidases in the ileum. In contrast, the activity of microvillus aminopeptidase and dipeptidyl peptidase IV in the ileum had a tendency to decrease.

Effect of neonatal androgen on the activity of peptidases in the rat brain inactivating luteinizing hormone-releasing hormone.

The activity of peptidase enzymes in different brain areas inactivating luteinizing hormone-releasing hormone (LH-RH) was investigated in normal male and female rats treated neonatally with androgen. Of the two fractions containing peptidase activity, changes were found only in the supernatant (soluble/cytoplasmic) fraction and were restricted to the hypothalamus and cortex. In both these brain areas, peptidase activity was considerably higher in androgen-treated than in normal female rats; there was no significant difference between activity in male rats and the androgen-treated female rats. These findings indicate that neonatal androgen produces masculinization of the mechanisms responsible for LH-RH inactivation in the hypothalamus which may reflect changes observed elsewhere in gonadotropin secretion in androgen-treated female rats. The changes in peptidase activity in the cortex may infer that sexual differentiation of the brain under the influence of androgen is not limited to the hypothalamus and can be manifested at extrahypothalamic sites within the central nervous system.