A comparative study of soluble calcium-dependent proteolytic activity in brain

Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains—the effects of a variety of protease inhibitors indicated that it is also a neurtral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class.     

Cysteine protease inhibitors reduce brain beta-amyloid and beta-secretase activity in vivo and are potential Alzheimer's disease therapeutics

Beta-secretase inhibitors that lower brain beta-amyloid peptides (Abeta) are likely to be effective for treating Alzheimer's disease (AD). Irreversible epoxysuccinyl cysteine protease inhibitors are known to reduce brain Abeta and beta-secretase activity in the guinea pig model of human Abeta production. In this study, acetyl-L-leucyl-L-valyl-L-lysinal (Ac-LVK-CHO) is also shown to significantly reduce brain Abeta and beta-secretase activity and brain Abeta in the same model. Ac-LVK-CHO is structurally distinct from the epoxysuccinyl inhibitors and is a reversible cysteine protease inhibitor. The results suggest that cysteine protease inhibitors generally, and reversible cysteine protease inhibitors specifically, have potential for development as AD therapeutics.     

Raft disorganization leads to reduced plasmin activity in Alzheimer's disease brains

The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.     

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.     

Defining a link between gap junction communication, proteolysis, and cataract formation

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.     

Identification and characterization of PEBP as a calpain substrate

Calpains are calcium- and thiol-dependent proteases whose dysregulation has been implicated in a number of diseases and conditions such as cardiovascular dysfunction, ischemic stroke, and Alzheimer's disease (AD). While the effects of calpain activity are evident, the precise mechanism(s) by which dysregulated calpain activity results in cellular degeneration are less clear. In order to determine the impact of calpain activity, there is a need to identify the range of specific calpain substrates. Using an in vitro proteomics approach we confirmed that phosphatidylethanolamine-binding protein (PEBP) as a novel in vitro and in situ calpain substrate. We also observed PEBP proteolysis in a model of brain injury in which calpain is clearly activated. In addition, with evidence of calpain dysregulation in AD, we quantitated protein levels of PEBP in postmortem brain samples from the hippocampus of AD and age-matched controls and found that PEBP levels were approximately 20% greater in AD. Finally, with previous evidence that PEBP may act as a serine protease inhibitor, we tested PEBP as an inhibitor of the proteasome and found that PEBP inhibited the chymostrypsin-like activity of the proteasome by approximately 30%. Together these data identify PEBP as a potential in vivo calpain substrate and indicate that increased PEBP levels may contribute to impaired proteasome function.     

The reconstituted 'humanized liver' in TK-NOG mice is mature and functional

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA’s proteolytic activity for mushroom defense against pathogens is discussed.     

Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Brain Regional Correspondence Between Alzheimer's Disease Histopathology and Biomarkers of Protein Oxidation

Abstract: Four biomarkers of neuronal protein oxidation [W/S ratio of MAL-6 spin-labeled synaptosomes, phenylhydrazine-reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)-demented and age-matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation-sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque-dense regions of the AD brain may represent environments of elevated oxidative stress.     

Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease

Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.     

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer's disease

Abnormal accumulation of neurotoxic beta-amyloid peptides (Abeta) in brain represents a key factor in the progression of Alzheimer's disease (AD). Identification of small molecules that effectively reduce brain levels of Abeta is important for development of Abeta-lowering agents for AD. In this study, we demonstrate that in vivo Abeta levels in brain are significantly reduced by the cysteine protease inhibitor E64d and the related CA074Me inhibitor, which inhibits cathepsin B. Direct infusion of these inhibitors into brains of guinea pigs resulted in reduced levels of Abeta by 50-70% after 30 days of treatment. Substantial decreases in Abeta also occurred after only 7 days of inhibitor infusion, with a reduction in both Abeta40 and Abeta42 peptide forms. A prominent decrease in Abeta peptides was observed in brain synaptosomal nerve terminal preparations after CA074Me treatment. Analyses of APP-derived proteolytic fragments showed that CA074Me reduced brain levels of the CTFbeta fragment, and increased amounts of the sAPPalpha fragment. These results suggest that CA074Me inhibits Abeta production by modulating APP processing. Animals appeared healthy after treatment with these inhibitors. These results, showing highly effective in vivo decreases in brain Abeta levels by these cysteine protease inhibitors, indicate the feasibility of using related compounds for lowering Abeta in AD.     

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.     

Protein Levels and Activity of Some Antioxidant Enzymes in Hippocampus of Subjects with Amnestic Mild Cognitive Impairment

Mild cognitive impairment (MCI) is generally referred to the transitional zone between normal cognitive aging and early dementia or clinically probable Alzheimer’s disease (AD). Most individuals with amnestic MCI eventually develop AD, which suggests that MCI may be the earliest phase of AD. Oxidative stress is observed in brain from subjects with both AD and MCI. Among others, two possibilities for elevated oxidataive stress are decreased activity or elevated expression of antioxidant enzymes, the latter as a response to the former. Accordingly, in the current study, the protein levels and activity of some antioxidant enzymes in the hippocampus of control and MCI brain were measured using Western blot analysis and spectrophotometric methods, respectively. Alterations in the levels and activity of a number of antioxidant enzymes in MCI brain compared to age-matched controls were found. These results are consistent with the hypothesis that oxidative stress may be an early event in the progression of amnestic MCI to AD. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Differential effects of calpain inhibitors on hypertrophy of cardiomyocytes

Two inhibitors of the calcium-dependent cysteine protease, calpain, have markedly different effects on the extent of hypertrophy induced by the alpha-adrenergic agonist, phenylephrine, of cultured neonatal rat ventricular myocytes. E64c, an inhibitor of calpain and other cysteine proteases, stimulated the hypertrophy by 59%. PD 150606, a specific calpain inhibitor, reduced the hypertrophy by 38%. Phenylephrine decreased the proteolysis of a calpain substrate by the cells 1–2 h after its addition but not at 24 h. PD 150606 inhibited proteolytic activity at all times, and the combination of phenylephrine and PD 150606 did not give greater inhibition. This suggests that cysteine proteases of the papain sub-family are involved with the hypertrophic response at two points, promoting hypertrophy at the first and limiting it at the second. Calpain appears to be the protease involved at the first point, and there may be another cysteine protease acting at the second site.     

Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change

Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.     

Network analysis of intrinsic functional brain connectivity in Alzheimer's disease

Functional brain networks detected in task-free (“resting-state”) functional magnetic resonance imaging (fMRI) have a small-world architecture that reflects a robust functional organization of the brain. Here, we examined whether this functional organization is disrupted in Alzheimer's disease (AD). Task-free fMRI data from 21 AD subjects and 18 age-matched controls were obtained. Wavelet analysis was applied to the fMRI data to compute frequency-dependent correlation matrices. Correlation matrices were thresholded to create 90-node undirected-graphs of functional brain networks. Small-world metrics (characteristic path length and clustering coefficient) were computed using graph analytical methods. In the low frequency interval 0.01 to 0.05 Hz, functional brain networks in controls showed small-world organization of brain activity, characterized by a high clustering coefficient and a low characteristic path length. In contrast, functional brain networks in AD showed loss of small-world properties, characterized by a significantly lower clustering coefficient (p<0.01), indicative of disrupted local connectivity. Clustering coefficients for the left and right hippocampus were significantly lower (p<0.01) in the AD group compared to the control group. Furthermore, the clustering coefficient distinguished AD participants from the controls with a sensitivity of 72% and specificity of 78%. Our study provides new evidence that there is disrupted organization of functional brain networks in AD. Small-world metrics can characterize the functional organization of the brain in AD, and our findings further suggest that these network measures may be useful as an imaging-based biomarker to distinguish AD from healthy aging.     

The 20S proteasome isolated from Alzheimer's disease brain shows post-translational modifications but unchanged proteolytic activity

Chronic neurodegenerative diseases are characterized by the accumulation of aggregated protein species, and functional impairment of the ubiquitin proteasome system has been hypothesized to contribute to neuronal cell loss. Decreased proteolytic activity of the 20S proteasome has been shown postmortem in crude brain lysates from Alzheimer's disease (AD) patients. In the present study, we demonstrate, however, that catalytic activity of the 20S proteasome increases during chromatographic purification from AD brains as compared with age-matched controls. By two-dimensional difference gel electrophoresis we detected pI shifts in several proteasome subunits in AD samples pointing to differential post-translational modifications. Moreover, we identified N-terminal acetylation and dephosphorylation of subunit alpha7 in AD by tandem mass spectrometry. Thus, reduced peptidase activity in AD brain extracts is not an intrinsic property of the 20S proteasome, but may be resulting from the presence of endogenous inhibitory proteins or substrates. Post-translational modifications of non-catalytic subunits in situ may contribute to the trend towards enhanced hydrolytic activity of the isolated 20S proteasome after removal of the endogenous inhibitors.