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Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease - PubMed

Review

Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease

Vivian Hook et al. Biochim Biophys Acta. 2012 Jan.

Abstract

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Figures

Figure 1
Figure 1. Structural Features of Proneuropeptides

Prohormone precursor protein structures indicate that active peptide neurotransmitters and hormones are flanked by multi-basic residues that represent sites of proteolytic processing to generate active neuropeptides. The precursor proteins are shown for preproenkephalin, preproopiomelanocortin, preproNPY (NPY, neuropeptide Y), preprodynorphin, preproCCK (CCK, cholecystokinin), and preprogalanin. The NH2-terminal signal sequence is known to be cleaved by signal peptidases at the RER (rough endoplasmic reticulum) and the resultant prohormone undergoes trafficking to Golgi apparatus and packaging into secretory vesicles where prohormone processing occurs.

Figure 2
Figure 2. Cathepsin L and Proprotein Convertase Pathways for Neuropeptide Production

Proneuropeptides typically contain active peptides flanked by paired basic residues. The dibasic processing sites undergo proteolytic cleavage at one of three sites (numbered 1, 2, and 3) which consist of cleavage at the NH2- or COOH-terminal sides of the dibasic residues, or between the dibasic residues. Peptide intermediates generated by cleavage at the NH2-terminal side of the dibasic site, or between the dibasic residues, will then require Arg/Lys aminopeptidase, represented by aminopeptidase B, to remove basic residues at the NH2-termini. Cleavage of proneuropeptides at the COOH-terminal side of dibasic residues then requires carboxypeptidase E to remove NH2-terminal basic residues [1, 5].

Figure 3
Figure 3. Cathepsin L is Identified as the Proenkephalin-Cleaving Activity in Secretory Vesicles
(a) E64-c cysteine protease inhibitor.

The cysteine protease inhibitor E64-c was found to be a potent inhibitor of the proenkephalin (PE) – cleaving activity in secretory vesicles isolated from adrenal medullary chromaffin cells of the sympathetic nervous system [2].

(b) Structure of DCG-04, an activity-based probe for cysteine proteases.

The modified cysteine protease inhibitor DCG-04 [2, 93], resulting from biotinylation of E64-c, was utilized for affinity-labeling of PE-cleaving activity in secretory vesicles.

(c) DCG-04 affinity labeling of cysteine protease activity in secretory vesicles.

DCG-04 affinity labeling of purified PE-cleaving activity reveals a 27 kDa protein band. This band was subjected to peptide sequencing by tryptic digestion and tandem mass spectrometry.

(d) Identification of purified PE-cleaving activity as cathepsin L by mass spectrometry for peptide sequencing

. Peptides derived from tryptic digests of DCG-04 affinity labeled 27 kDa proteins, sequenced by CID (MS/MS) mass spectrometry, are illustrated as the underlined amino acid sequences of bovine cathepsin L.

Figure 4
Figure 4. Localization of cathepsin L within enkephalin-containing secretory vesicles
(a) Cathepsin L colocalization with (Met)enkephalin (ME) by immunofluorescence confocal microscopy

. Immunofluorescence localization of cathepsin L (cat. L) was assessed by anti-cathepsin L detected with anti-rabbit IgG-Alexa 488 (green fluorescence), and ME was detected with anti-ME and anti-mouse IgG-Alexa 594 (red) [2]. Colocalization is illustrated by overlay of the images, illustrated by yellow fluorescence. Nuclei are illustrated by DAPI blue staining.

(b) Immunoelectron microscopy demonstrates colocalization of cathepsin L and (Met)enkephalin in secretory vesicles.

Cathepsin L in secretory vesicles was indicated by anti-cathepsin L detected with 15 nm colloidal gold conjugated anti-rabbit IgG, and ME was detected with anti-ME and 6 nm colloidal gold conjugated to anti-mouse IgG. The presence of both 15 and 6 nm gold particles within these vesicles demonstrated the in vivo colocalization of cathepsin L and ME.

Figure 5
Figure 5. Aβ production in the regulated secretory pathway of neurons provides the majority of extracellular Aβ that causes memory loss

Aβ peptides are generated by proteolytic processing of the amyloid precursor protein (APP) in secretory vesicles that undergo axonal transport from the neuronal cell body to nerve terminals, where Aβ is secreted. Secretory vesicles of the regulated secretory pathway (yellow circles) provide the majority of secreted, extracellular Aβ peptides [, , , –76]. Some Aβ is also provided by the basal, constitutive secretory pathway (constitutive secretory vesicles shown as blue circles). Intracellular production of Aβ within secretory vesicles occurs by cleavage at the N-terminus of Aβ within APP, achieved by proteases known as β-secretases, and cleavage at the C-termini of Aβ within APP which is achieved by γ-secretases. Proteolytic processing by β- and γ-secretases results in Aβ peptides of 40 and 42 residues, known as Aβ(1–40) and Aβ(1–42). Extracellular Aβ peptides in brain accumulate as oligomers and aggregates in amyloid plaques, and cause loss of memory in Alzheimer’s disease.

Figure 6
Figure 6. Model of APP and protease interactions at the β-secretase site
a. Interactions of amyloid precursor protein (APP) with proteases at the β-secretase site for production of neurotroxic β-amyloid peptides (Aβ).

The APP precursor protein undergoes proteolytic cleavages at the β-secretase site and the γ-secretase sites to generate Aβ peptides, consisting primarily of Aβ(1–40) and Aβ(1–42) that contain the same N-terminus with differences in their C-termini. The primary sequence of Aβ peptides and flanking residues at the secretase cleavage sites are illustrated.

b. Protease active site interactions with APP at the β-secretase site: Schechter and Berger model.

(i) The active site of the enzyme is composed of several subsites. The scheme shows an active site of six subsites, termed S1 to S3 and S1' to S3'. Subsites are located on both sides of the catalytic site and are numbered from this point in either direction. The positions of amino acid residues of the hexapeptide substrate are counted from the point of cleavage and thus have the same numbering as the subsites they occupy (P1 to P3 and P1' to P3'). Cleavage occurs between P1 and P1' [85, 86]. (ii) Wild-type β-secretase cleavage site: P3 to P3’ residues. Cleavage of the wild-type site β-secretase site of APP occurs between Met-↓Asp which represent the P1-P1’ residues. The Val-Lys-Met residues represent the P3, P2, and P1 residues, respectively; the Asp-Ala-Glu residues are the P1’, P2’, and P3’ residues, respectively.

Figure 7
Figure 7. Cathepsin B is identified as a β-secretase in regulated secretory vesicles
a. Purification of proteolytic activity cleaving the wild-type β-secretase cleavage site substrate Z-Val-Lys-Met-MCA: active-site directed probe labeling

. Regulated secretory vesicles of neuronal-like chromaffin cells (from the sympathetic nervous system) produce and secrete Aβ peptides [9, 10]. Purification of proteolytic activity that cleaves the wild-type β-secretase site, present in the majority of Alzheimer’s patients, was conducted using isolated secretory vesicles of the regulated secretory pathway. The β-site cleaving protease was labeled with the active-site directed affinity probe DCG-04 (shown by the arrow) as a protein band of ~ 31 kDa (lane 1). The selective inhibitor of cathepsin B, CA074, blocked the DCG-04 probe labeling, suggesting that the protease may be represented by cathepsin B (lane 2) [10]

b. Inhibition of Z-Val-Lys-Met-MCA cleaving activity by E64c and CA074

. The Z-Val-Lys-Met-MCA cleaving activity purified from Aβ-containing regulated secretory vesicles (chromaffin secretory vesicles) was inhibited by the cysteine protease inhibitor E64c and by the selective inhibitor of cathepsin B, CA074 [10].

c. Peptide sequencing identifies cathepsin B as β-secretase.

The 31 kDa band (shown in part ‘a.ii) was indicated as the responsible enzyme for β-secretase activity in regulated secretory vesicles. The 31 kDa band was subjected to peptide sequencing by tryptic digestion and tandem mass spectrometry (MS/MS) of peptide fragments. The determined sequences of tryptic peptides are underlined within the complete primary sequence of cathepsin B [10]

d. Colocalization of cathepsin B with Aβ peptides in regulated secretory vesicles: analyses by immunoelectron microscopy.

Cathepsin B (Cat. B, 15 nm gold particles) was colocalized with Aβ40 (6 nm gold particles) within regulated secretory vesicles isolated from neuronal chromaffin cells (panel i). Cathepsin B (15 nm gold particles) was also colocalized with Aβ42 (6 nm gold particles) in regulated secretory vesicles (panel ii) [10].

Figure 8
Figure 8. Inhibitors of cathepsin B reduce Aβ production in regulated secretory vesicles

The effects of cathepsin B inhibitors, CA074 (10 µM, ◆) and CA074Me (10 µM, □), on the production of Aβ40 from endogenous APP in regulated secretory vesicles isolated from chromaffin cells was evaluated in time course studies [10]. Controls without inhibitors (●), or with the cysteine protease inhibitor E64c (10 µM, ○) were included. Each inhibitor was tested in triplicate; values represent×± sem.

Figure 9
Figure 9. The inhibitors CA074Me or E64d improve memory deficit in London APP mice assessed by the Morris water maze test

Memory function was assessed in the Morris water maze test after administration of CA074Me or E64d by icv administration [13]. CA074Me is a prodrug form of CA074 (conversion to CA074 by cellular esterases), a specific inhibitor of cathepsin B. E64d is a prodrug form of E64c (conversion to E64c by cellular esterases). The latency period measures the time it takes the animal to swim to a submerged platform after training, with shorter times reflecting improved memory. Results are displayed as the mean latency period (seconds) ± standard deviation (SD), with statistical significance indicated (*** p < 0.0001, student’s t-test). The latency time for wild-type, normal mice is illustrated by the dotted line. Results show that treatment with the inhibitors improves the memory deficits towards normal memory function of wild-type mice.

Figure 10
Figure 10. The inhibitors CA074Me or E64d reduce brain Aβ peptides and CTFβ derived from APP by β-secretase
a. Reduction of brain Aβ40 after treatment of London AD mice with CA074Me or E64d

. London APP mice were treated with CA074Me or E64d, and Aβ40 levels in brain were measured by ELISA assays and expressed as percent of the control Aβ40 levels. Results are expressed as the mean (% control) ± SD, with statistical significance indicated (*** p < 0.0001, student’s t-test). Brain levels of Aβ40 are substantially reduced after treatment with the inhibitors [13].

b. Reduction of brain Aβ42 after treatment of London AD mice with the inhibitors.

After treatment of London APP mice with CA074Me or E64d, Aβ42 levels in brain were measured by ELISA assays and expressed as the percent of the control Aβ42. Results are expressed as the mean (% control) ± SD, with statistical significance indicated (*** p < 0.0001, student’s t-test). Brain levels of Aβ42 are decreased after treatment with the inhibitors [13].

c. Reduced CTFβ in brain after inhibitor treatment.

Analyses of CTFβ derived from APP by β-secretase in London APP mouse brains were performed with brain extracts, and the CTFβ band (12 kDa) was quantitated by densitometry. Results show a decrease in CTFβ after inhibitor treatment [13]. Results are expressed as the mean (% control) ± SD, with statistical significance indicated (*** p < 0.0001, student’s t-test).

d. Proteolytic processing of APP into CTFβ and Aβ peptides by β- and γ-secretases

. Cleavage of APP at the β-secretase site generates the C-terminal β-secretase fragment (CTFβ). Processing of CTFβ by γ-secretase results in the generation of Aβ peptides.

Figure 11
Figure 11. Knockout of the cathepsin B gene in transgenic mice expressing human wild-type APP (hAPPwt) reduces brain Aβ40 and Aβ42
a. Aβ40 levels in brain

. The wild-type Cat B+/+ and knockout Cat B−/− mice expressing human wild-type APP (hAPPwt) contained brain Aβ40 levels of 40.0 ± 8.4 and 13.6 ± 3.0 nM, respectively (significant, **p < 0.007). Knockout of the cathepsin B gene resulted in a 66% reduction in brain Aβ40 in mice expressing hAPPwt [14].

b. Aβ42 levels in brain

. The Cat B+/+ and Cat B−/− mice expressing hAPPwt contained brain Aβ42 levels of 4.8 ± 1.0 and 1.5 ± 8.4 nM, respectively (significant, **p < 0.007). Knockout of the cathepsin B gene resulted in a 68% reduction in brain Aβ42 in animals expressing hAPPwt [14].

Figure 12
Figure 12. The secretory vesicle proteome for biosynthesis and secretion of active peptides

Proteins of the secretory vesicle, known as the neuroproteome, participate in the biosynthesis, storage, and regulated secretion of active peptides, as described in this review article. Thus, the active peptides are generated and secreted by the proteome of regulated secretory vesicles. The secretory vesicle proteome consists of soluble and membrane proteins that participate in secretory vesicle functions for providing neuropeptides for cell-cell communication in the nervous and endocrine systems. Proteomic studies of the soluble and membrane fractions of neuropeptide secretory vesicles isolated from adrenal medullary chromaffin cells of the sympathetic nervous system (bovine) indicate the protein systems participating in production of neuropeptides and β-amyloid for regulated secretion that include neuropeptides and neurohumoural factors, proteases, neurotransmitters enzymes and transporters, receptors, enzymes, carbohydrate functions, lipids, reduction-oxidation, ATPases and nucleotide metabolism, protein folding, signal transduction and GTP-binding proteins, vesicular trafficking and exocytosis, structural proteins, and cell adhesion proteins [111, 112].

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