Quantitative determination and localization of cathepsin D and its inhibitors.

Abstract

Cathepsin D (EC 3.4.23.5) is a lysosomal aspartyl endopeptidase, localized in all cells and tissues, except for mature erythrocytes [1][2][3]. Methods used to determine the activity, concentration and cellular distribution of cathepsin D, but not its inhibitors, have previously been the subject of literature reports [4][5][6][7][8][9][10][11][12]. However, since the time of their publication a number of new substrates and analytical techniques have been implemented. Structure, specificity, mechanism of actionCathepsin D is synthesized in the rough endoplasmic reticulum as preprocathepsin D, built up of 412 amino acid residues [13][14][15]. As a result of cleavage of the 20-amino acid signal prepeptide, it is converted into procathepsin D which undergoes glycosylation and disulphide bridges are formed in its molecule. Procathepsin D is transported from cisterns of the rough endoplasmic reticulum to the Golgi apparatus, from which, with the involvement of mannoso-6-phosphate (M-P-6) receptors, it is transferred to primary lysosomes [16,17]. As the M-6-P receptors are known to occur in the primary lysosomes but not in the mature ones, they can be used to distinguish between these two types of lysosomes [18]. In the acidic environment of the lysosomes (pH 4.5-5.5), due to autocatalytic cleavage of the 44-amino acid propeptide from the N-terminal molecule, procathepsin D is converted into the active one-chain form. The actions of cysteine proteinase, aminopeptidases and carboxypeptidases lead to the formation of an active two-chain form of cathepsin D (Fig. 1). These chains are bound by hydrophobic bonds. The molecular weight of the ultimate mature form of cathepsin D is 48 (14+34) kDa. The proteolytic activities of the one-chain and two-chain forms are very similar [19,20]. Modification of the polypeptide chain, different oligosaccharide composition types and phosphorylation/dephosphorylation in the amino saccharide residues contribute to marked molecular heterogenicity of cathepsin D and cause differences in isoelectric points of the respective isoenzymes between pH 4.5 -6.5 [21,22].The use of peptides with the known primary structure allows identification of amino acid residues that form peptide bonds cleaved by cathepsin D. For this purpose, synthetic peptides [23] and chains A and B of bovine insulin can be used (Fig. 2). Cathepsin D cleaves the peptide bonds found within the polypeptide chain, formed by carboxyl groups of the hydrophobic amino acid residues: aromatic -trypto-

Lysosomal function in macromolecular homeostasis and bioenergetics in Parkinson's disease

“…The cleavage of proenzymes into active forms occurring within the greater lysosomal systems prevents premature activation of hydrolases outside of the lysosomes. Endogenous inhibitors of some of the hydrolases also exist to facilitate the compartmentalized activity of the lysosomal enzymes [103-106]. Intracellular metabolites may be upregulated in response to stress to modulate lysosomal enzyme activities [107].…”

Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions

“…As an inhibitor of aspartic acid proteases, the IC 50 values of pepstatin are 15 lM, <5 nM and <40 nM for human renin, pepsin and cathepsin D, respectively [57,58]. The most specific inhibition of cathepsin D, according to literature, is obtained by using an anti-cathepsin D antibody [53].…”

“…Cathepsin D is a lysosomal aspartyl endopeptidase that resides in all cells except erythrocytes [53]. Similarly, cathepsin E is an intracellular aspartyl protease of the endo-lysosomal pathway [54].…”

“…Hence, neither of them should be present in plasma in high amounts under physiological conditions. Pepstatin A is a potent inhibitor for cathepsin D [53,55,56], but inhibits also other proteases such as pepsin and renin. As an inhibitor of aspartic acid proteases, the IC 50 values of pepstatin are 15 lM, <5 nM and <40 nM for human renin, pepsin and cathepsin D, respectively [57,58].…”

Influence of thrombophlebitis on TGF-β1 and its signaling pathway in the vein wall.

“…However, when comparing varicose veins and varicose veins complicated by thrombophlebitis, a significant increase in active TGF-β1 expression was demonstrated in the course of thrombophlebitis. A number of proteases, including matrix metalloproteinases (e.g., MMP-9) and cathepsin D, are capable of activating latent TGF-β1 [12,[28][29][30]. It is noteworthy, that increased MMP-9, as well as cathepsin D activities have been demonstrated in varicose veins complicated by thrombophlebitis [31,32].…”