Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Quantitation of oxidative damage to tissue proteins.

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl-acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol-ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1-4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.

Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis.

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Effects of tricyclic compounds on membrane binding of bivalent cations, activities of acetylcholinesterase and some tissue proteases.

A tricyclic compound tetrahydroaminoacridine is known to improve the cognitive function in Alzheimer's disease. The possible mechanism of action of acridine and structurally related tricyclic compounds was studied on the bivalent cation content of bacterial membrane, rat brain acetylcholinesterase and some tissue proteases in model experiments. Acridine orange and disubstituted chlorpromazine (CPZ) derivatives lowered Ca2+ and Mg2+ binding and membrane polarization in the simplest biological membrane (E. coli), as revealed by reactor neutron activation analysis. Acetylcholinesterase (AChE) was inhibited by CPZ, 3,7,8-trihydroxy-CPZ, acridine orange partially saturated desipramine, imipramine, trans-clopenthixol and tetrahydrocannabidiolic at 10(-4) to 10(-5). A metalloproteinase, MMP-7-ase, was inhibited by tetrahydrocannabidiolic acid, 3,7,8-trihydroxy-CPZ, acridine orange but other tissue proteinases, ATN-ase and cathepsin B, were less sensitive to these compounds. (ATN-ase is an acetyltyrosine-p-nitroanilide splitting enzyme, a serine protease). The chelate complex forming ability and electron donor capacity of the compounds may play a role in the biological effects tested. It is assumed that compounds which do not displace bivalent cations in membranes may exert an inhibitory effect on AChE, and that metalloproteinase enzymes may be promising for the treatment of degenerative brain diseases.

Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.

Regulation of proteolytic enzyme activities in muscles practice and hypothesis.

Two weeks of immobilization in shortened state caused 50% decrease in muscle mass and an increase in lysosomal proteinase activities. In this study causes of elevated protease activities in muscle are studied. Many factors could play a role in these elevated proteinase activities. We have found that redox state, ATP content, fuel supply and glucocorticoid receptor number were important in this period. Testosterone, insulin or proteinase inhibitors were not proved to play role in elevated proteinase activities. These practical results are explained by the results achieved in other types of cells. We conclude that changes in redox potential and/or oxygen free radical content of muscle elements can cause a post-translational covalent modification of cysteine proteinases and -SH dependent metalloproteinases, leading thereby to their activation. Lysosomal cysteine proteinases can activate procathepsin D that can damage lysosomal cysteine proteinase inhibitors and in another path it activates procathepsin B, L and H reversely. This feed-back regulation and the activation of cysteine proteinases by metalloproteinases might accelerate the proteinase activities in skeletal muscles.

Soluble metalloendopeptidase (MMP-7ase) activity in mouse kidney cytosol.

Previously, MMP-7ases were isolated from rat skeletal muscle by gel filtration and anion exchange chromatography. The enzyme that hydrolyzed succinyl-Ala-Ala-Pro-Phe-AMC (AMC: 7-amino-4-methyl-coumarin) was inhibited by EDTA. In this study we attempted to isolate MMP-7ase from mouse kidney. The isolation procedure was the same as that previously used for skeletal muscle. Kidneys of ICR mice were homogenized and, after centrifugation, the supernatant fraction was subjected to gel filtration chromatography. The fraction with the highest activity (Mr 67-72 kDa) was subjected to anion exchange chromatography, which showed three peaks of activity. The second peak hydrolyzed succ-Ala-Ala-Pro-Phe-AMC, but had low activity against Arg- or Ala-AMC. This peak was a single protein (Mr 68-72 kDa) and its activity could be inhibited with EDTA. Several tri- and tetrapeptide derivatives were tested as substrates for this enzyme and the best was found to be succ-Ala-Ala-Pro-Phe-AMC. We can conclude that mouse kidney cytosol contains a metalloendopeptidase similar to muscle MMP-7ase.

Proteolytic enzyme activities in rat peritoneal exudate.

Proteinase activities in rat thioglycollate elicited peritoneal cells and the cell-free supernatant (lavage fluid) were measured by using the following substrates: Suc-Ala-Ala-Pro-Phe-Methyl-Coumarin-Amide (for cathepsin G or chymase), Suc-Ala-Ala-Ala-AMC (for elastase or elastase-like), Z-Arg-Arg-AMC (for cathepsin B), haemoglobin (for cathepsin D) and Ala-AMC (for alanine-aminopeptidase: AAP). The enzyme activities were correlated to the quantitative distribution of various cell types in the exudate from 0 to 192 nd h. In the supernatant all the examined activities showed a higher value at 72nd h. In the cells activity of chymase and AAP proved to be very high at 0 h but after four h the activities were dropped. From this time all enzyme activities started to elevate till the 24th h. At the 96th h only the activity of cathepsin B and AAP had a high value. We conclude that the intracellular activation and secretion of proteolytic enzymes characteristic for the various peritoneal cell types involved in the acute and chronic inflammatory reaction can be followed by activity measurements using enzyme-specific substrates and inhibitors.

Regulation of proteinase activation in mammalian tissues.

A lot of new results have been published on activation of one or more proteinases in mammalian tissues. Explanation of proteinase activation without invasion of leukocyte during atrophy of mammalian tissues is the subject of the present study. The mechanism of activation process can be divided into two parts. The first one consists of modification of proteinases and their inhibitors by oxidation or reduction including oxygen free radical reactions. The second part of activation is a cascade or cycle including limited proteolysis of proenzymes and/or proteinase inhibitors. The hypothetic system of proteinase activation can give a hope for regulation of proteinase activity in mammalian tissues under pathological- or pathologic-like conditions as during exhausting exercise.

Activity of the lysosomal cysteine proteinases (cathepsin B,H,L) and a metalloproteinase (MMP-7-ase) in the serum of cystic fibrosis homozygous children.

Lysosomal cysteine proteinase (cathepsin B, H, L) and metalloproteinase (MMP-7-ase) activities were measured from serum of 19 cystic fibrosis (CF) homozygotes and of 13 healthy children, as control group. The activity of cathepsin B and H significantly increased in the CF-group.

Correlation of the fibrinogen level with blood pressure and plasma renin activity in rats with early Goldblatt hypertension.

Goldblatt hypertension was induced in rats by constricting the renal artery on one side. In one group of animals the contralateral kidney remained untouched (two-kidney hypertension), while in the other it was removed (one-kidney hypertension). In the two-kidney hypertension group, renin activity was higher than in the control animals, the fibrinogen was normal both in arterial and venous blood while in one-kidney hypertension the PRA was normal, but the fibrinogen was increased. A close significant correlation could be demonstrated between blood pressure and fibrinogen.

Cysteine and metalloproteinase activities in serum of Duchenne muscular dystrophic genotypes.

Lysosomal cysteine proteinase (cathepsin B, H, and L) and MMP-7ase muscle metalloproteinase activities were measured in serum from Duchenne muscular dystrophic male patients and their mothers as gene-carriers. The activity of cathepsin H significantly increased in the Duchenne muscular dystrophic (DMD)-hemizygotes group and in the group of DMD heterozygotes. Significant positive correlation was found between the activity of serum creatine kinase (which previously has been proven to be a marker of muscular dystrophy) and of cathepsin L in the DMD-hemizygotes group. Furthermore, correlations were found between the activity of creatine kinase and MMP-7ase or between activity of creatine kinase and cathepsin H in the DMD heterozygotes. The changes in activity of proteolytic enzymes in serum of dystrophic patients can be explained by the elevated proteolytic enzyme activity in dystrophic muscle observed previously.

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH.

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.

Experimental investigations on hypokinesis of skeletal muscles with different function. IV. Changes in the sarcoplasmic proteins.

The changes in the sarcoplasmic proteins of the m. gastrocnemius and m. soleus were examined by biochemical methods on the 5th, 7th, 14th and 28th days after plaster cast immobilization of the right hind limbs of adult rabbits. During 4 weeks the soluble/myofibrillar protein ratio increased from 0.47 to 0.75 in the m. gastrocnemius, and to 0.85 in the m. soleus. Evaluation of the relative quantities of the components identified after gel-electrophoresis separation led to the following results: (1) There was no, or no appreciable change in the glyceraldehyde-3-phosphate dehydrogenase, creatine kinase and enolase activities. (2) The enzymes lactate dehydrogenase, aldolase and the glycogenolytic enzymes showed a relative decrease in both muscles. (3) Phosphoglycerate kinase, phosphoglucose isomerase and pyruvate kinase increased in both muscles. (4) Changes of opposite directions were exhibited by myoglobin, myokinase and F-protein. These results provide new data on the biochemical characterization of these functionally different muscles, and on the mechanism of disuse atrophy.

Stretch-induced prostaglandins and protein turnover in cultured skeletal muscle.

Intermittent repetitive mechanical stimulation of differentiated avian skeletal muscle cells in vitro for 48 h stimulates skeletal muscle growth [Am. J. Physiol. 256 (Cell Physiol. 25): C674-C682, 1989]. During the first 2-3 h of stimulation, temporary muscle damage occurs based on increases in creatine kinase efflux, total protein degradation rates, and several proteinase activites. With continued mechanical stimulation for several days in serum-containing medium, the proteinase activities return to control levels, and total protein degradation rates decrease to levels less than static controls. Decreased protein degradation thus contributes to stretch-induced cell growth. The efflux of prostaglandins (PG) E2 and F2 alpha but not 6-keto-PGF1 alpha increase with mechanical stimulation. During the first 5 h of stimulation, PGE2 and PGF2 alpha efflux rates increase 101 and 41%, respectively. PGE2 efflux returns to control levels by 24 h of mechanical stimulation, whereas PGF2 alpha efflux is continuously elevated (41-116%) for at least 48 h. The long-term stretch-induced elevation of PGF2 alpha efflux correlates with a 52-98% long-term increase in total protein synthesis rates. The prostaglandin synthesis inhibitor indomethacin partially blocks early stretch-induced cell damage and long-term stretch-induced cell growth. The results indicate that both of these processes are partially dependent on stretch-induced increases in prostaglandin synthesis.

Calcium-dependent proteolysis of calcium-binding proteins.

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.