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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 has been demonstrated 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 contrasts 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 function. 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.
Cathepsin proteases participate in protein degradation in lysosomes, resulting in inactivation of protein functions. However, new research findings indicate novel biological functions of cathepsins in secretory vesicles. The novel secretory vesicle role of the cysteine protease cathepsin L has been demonstrated for production of active peptides required for cell-cell communication in the nervous and endocrine systems [1–8]. Significantly, cathepsin B in secretory vesicles produces neurotoxic β-amyloid (Aβ) [9–16] that is known to be a major factor involved in the development of Alzheimer’s disease (AD) that results in severe memory loss [17–19]. Inhibitors of cathepsin B result in reduced brain levels of Aβ peptides [11–16] and substantial improvement in memory deficits in an Alzheimer’s mouse model [13, 15], indicating cathepsin B as a logical drug target for AD. This review article explains the interdisciplinary strategies in cell biology, chemical biology, and genetics utilized for discovery of new biological functions of secretory vesicle cathepsins L and B for the biosynthesis of active peptides. Furthermore, the secretory vesicle proteome that supports these functions of cathepsins L and B have been investigated to gain insight into the unique intravesicular environment for production of active peptides by these cysteine cathepsin proteases.
Secretory vesicles in neurons and endocrine cells provide regulated secretion of biologically active small peptides for cell-cell communication. Peptides comprise the majority of neurotransmitters and hormones among physiological systems. These peptide neurotransmitters and hormones are first synthesized as proprotein precursors that must undergo limited proteolysis to generate active peptides. Proteolytic processing of the proproteins occurs primarily within the secretory vesicle organelle and, thus, the search for such proteases has recently identified the cysteine protease cathepsin L as a key processing enzyme for production of numerous peptide neurotransmitters [1–8]. Cathepsin L produces substantial portions of brain peptide neurotransmitters, known as neuropeptides, that include enkephalin, NPY (neuropeptide Y), dynorphins, cholecystokinin, and others as demonstrated by in vivo cathepsin L gene knockout and cellular cathepsin L gene expression studies. The chemical biological and genetic strategies utilized to identify cathepsin L as a proneuropeptide processing enzyme in secretory vesicles is covered by this review.
Peptide neurotransmitters are essential for activity-dependent neurotransmission in the nervous system. Many neuropeptides also function in peripheral systems for endocrine regulation of physiological functions. Moreover, the nervous and endocrine systems communicate with one another via these neuropeptides.
Production of neuropeptides requires proteolytic processing of their precursor proteins to result in a multitude of distinct peptides with diverse physiological actions such as enkephalin for opioid peptide regulation of analgesia [20, 21], ACTH induction of steroid synthesis , galanin regulation of cognition , neuropeptide Y regulation of feeding behavior [24, 25], and numerous other functions (Table 1). The primary structures for proneuropeptides indicate that neuropeptides are typically flanked at their NH2- and COOH-termini by pairs of basic residues, and sometimes by monobasic residues [5, 26, 27] (Figure 1). These multi-basic and monobasic residues are cleaved to generate the active neuropeptides. Clearly, proteolytic pathways represent key steps for the biosynthesis of essential peptide neurotransmitters and hormones.
Proteolytic processing of proneuropeptides may occur at one of three positions at paired basic processing sites. These cleavages may consist of processing at the COOH- and NH2-termini of the dibasic residues, or between the dibasic residues (Figure 2). Processing at the dibasic residue sites is known to be achieved by two distinct pathways consisting of the recently discovery cathepsin L cysteine protease pathway combined with the well known subtilisin-like prohormone convertase pathway utilizing primarily PC/13 and PC2 prohormone convertase members of the PC family [5, 26, 27] Resultant peptide intermediates require removal of basic residues from COOH- and/or NH2-termini by carboxypeptidase and aminopeptidase enzymes, respectively.
The strategy to elucidate the major proneuropeptide-cleaving activity in secretory vesicles was to determine the protease subclass for activity cleaving proenkephalin and then to identify the responsible protease(s) by activity-probe labeling and mass spectrometry . Neuropeptide secretory vesicles of the sympathetic nervous system in chromaffin cells were utilized for purification of the proenkephalin cleaving activity. The activity was substantially inhibited by selective inhibitors of cysteine proteases [1, 2].
Chemical biology has developed sophisticated activity-based probes for identification of protease and enzyme families . Activity-based profiling of active cysteine proteases was instrumental for identification of the protease responsible for proenkephalin cleaving activity in chromaffin granules. The activity probe DCG-04, the biotinylated form of E64c that inhibits cysteine proteases, was utilized for specific affinity labeling of the the proenkephalin cleaving enzyme activity (Figure 3a–c) . Two-dimensional gels resolved DCG-04 labeled proteins which were identified as cathepsin L by mass spectrometry (Figure 3d). These findings suggested a new biological function for cathepsin L in secretory vesicles for producing neuropeptides.
The identification of cathepsin L in secretory vesicles indicated the novel localization of cathepsin L in this organelle. Confirmation of the localization of cathepsin L within secretory vesicles was demonstrated by immunofluorescence confocal microscopy and immunoelectron microscopy (Figure 4) [2, 3]. Furthermore, cathepsin L undergoes cosecretion with enkephalin from regulated secretory pathway . Cellular routing and trafficking of cathepsin L was demonstrated by coexpression of cathepsin L with proenkephalin in neuroendocrine PC12 cells (derived from rat adrenal medulla). Expression of cathepsin L resulted in its trafficking to secretory vesicles that contain enkephalin and neuropeptides . These findings indicated the novel location of cathepsin L in neuropeptide-containing secretory vesicles.
Cathepsin L cleaves at dibasic and monobasic residue sites of proneuropeptides. Endogenous secretory vesicle cathepsin L cleaves proenkephalin (PE) at such dibasic residue sites. Cathepsin L generated (Met)enkephalin from the PE-derived intermediate BAM-22P via cleavage at the dibasic ↓Arg-↓Arg and monobasic ↓Arg sites, and the PE-derived peptide F intermediate was cleaved by cathepsin L at dibasic ↓Lys-↓Lys and ↓Lys-Arg sites [2, 30]. Further cleavage studies with peptide-MCA substrates showed that cathepsin L cleaves at the COOH-terminal side of the dibasic sites, as well as at the N-terminal side of basic residues . Cathepsin L generates peptide intermediates with basic residue extensions at NH2- and COOH-termini, which are then removed by aminopeptidase B and carboxypeptidase E exopeptidases, respectively (Figure 2). These exopeptidases have been shown to participate in neuropeptide biosynthesis in secretory vesicles [5, 32–34]. These basic residue cleavage specificities of cathepsin L are appropriate for processing proprotein precursors into active neuropeptides.
The in vivo role of cathepsin L in enkephalin peptide production was assessed in cathepsin L gene knockout mice. Brain levels of (Met)enkephalin (ME) were reduced by ~ 50% in cathepsin L knockou mice compared to wild-type controls . Brains contained a higher ratio of proenkephalin/ME in brain, indicating retarded proenkephalin processing. Thus, the knockout results demonstrate the in vivo function of cathepsin L for enkephalin neuropeptide production.
Studies of cathepsin L expression showed that cathepsin L participates in cellular processing of proenkephalin into (Met)enkephalin . Cathepsin L generated high molecular weight PE-derived intermediates (of ~23, 18–19, 8–9, and 4.5 kDa) that were similar to those in vivo in chromaffin granules . Such results demonstrated a cellular role for cathepsin L in the production of (Met)enkephalin in secretory vesicles.
Continued investigation of cathepsin L in secretory vesicles demonstrated its prominent role in the biosynthesis of numerous neuropeptides represented by neuropeptide Y (NPY), POMC-derived ACTH, β-endorphin, and α-MSH, as well as dynorphin, CCK, and catestatin neuropeptides [3, 4, 6–8]. Illustration of the function of cathepsin L for producing neuropeptides has been demonstrated by studies of cathepsin L protease gene knockout (summarized in Table 2), cathepsin L expression, and small molecule inhibition of cathepsin L.
NPY in the brain functions as a peptide neurotransmitter for the regulation of feeding behavior [24, 25]. In the peripheral sympathetic nervous system, NPY regulates blood pressure . Novel findings show that cathepsin L participates as a key proteolytic enzyme for NPY production . NPY in cathepsin L knockout (KO) mice was significantly reduced in brain and adrenal medulla by 80% and 90%, respectively. Cathepsin L is colocalized with NPY in secretory vesicles of brain cortical neurons and in chromaffin cells of adrenal medulla. Coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY via cleavage of proNPY at paired basic residues. In contrast, PC2 gene knockout mice show no change in NPY brain levels  but a peptidomic study showed a partial decrease in NPY . The field has not yet reported studies of NPY in PC1/3 knockout mice. These studies of demonstrate an important role for cathepsin L in the production of NPY.
Production of the pituitary hormones ACTH, β-endorphin, and αMSH utilize cathepsin L for processing POMC (proopiomelanocortin) . ACTH stimulates glucocorticoid synthesis in adrenal cortex, β-endorphin is an endogenous opioid for analgesia, and α-MSH is involved in pigmentation of the skin. Cathepsin L knockout (KO) mice have major decreases in ACTH, β-endorphin, and α-MSH that are reduced to 23%, 18%, and 7%, respectively, of wild-type controls (100%) in pituitary. Increased levels of POMC in these knockout mice are consistent with cathepsin L processing of POMC. Cathepsin L is colocalized with β-endorphin, α-MSH, and ACTH in pituitary secretory vesicles. Cathepsin L is also partially colocalized with the lysosomal marker lamp-1 in pituitary. Expression of cathepsin L in pituitary AtT-20 cells increased ACTH and β-endorphin production. Treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced ACTH and accumulation of POMC. For comparison, PC2 knockout (KO) mice showed substantial reduction in α-MSH levels in pituitary and brain , indicating that cathepsin L and PC2 function jointly for α-MSH production. ACTH and β-endorphin were elevated in pituitaries of PC2 KO mice suggesting that ACTH and β-endorphin serve as substrates for PC2. These studies demonstrate a prominent role for cathepsin L, combined with PC2, in the production of ACTH, β-endorphin, and α-MSH peptide hormones in secretory vesicles.
Dynorphin opioid neuropeptides mediate neurotransmission for analgesia and behavioral functions [39–41]. Dynorphin A, dynorphin B, and α-neoendorphin are generated from prodynorphin. Cathepsin L participates in producing dynorphins from prodynorphin . Cathepsin L KO mouse brains have decreased levels of dynorphin A, dynorphin B, and α-neoendorphin that are reduced by 75%, 83%, and 90%, respectively, compared to controls. Cathepsin L in brain cortical neurons is colocalized with dynorphins in secretory vesicles. Cellular coexpression of cathepsin L with prodynorphin in PC12 cells results in production of dynorphins A and B. Comparative studies of PC1/3 and PC2 convertases show that PC1/3 KO mouse brains had a modest decrease in dynorphin A, and PC2 knockout mice showed a minor decrease in α-neoendorphin . These results demonstrate a prominent role for cathepsin L, jointly with PC1/3 and PC2, for production of dynorphin neuropeptides.
CCK is a neurotransmitter whose production utilizes cathepsin L for processing the proCCK precursor to generate active CCK8 neuropeptide in brain . In cathepsin L knockout (KO) mice, CCK8 levels are reduced in brain cortex by ~75%. PC1/3 or PC2 KO mice showed more modest decreases of CCK8 [42–44]. Therefore, data from the cathepsin L KO results provide strong evidence for cathepsin L as a key protease responsible for CCK8 production in brain.
The catestatin peptide secreted in the adrenal medulla of the sympathetic nervous system regulates blood pressure and stress [45, 46]. Catestatin is generated from the precursor chromogranin A (CgA) by cathepsin L . Cathepsin L colocalizes with CgA in the secretory vesicles. Cathepsin L cleaves CgA in vitro to generate catestatin-related peptides. Thus far, PC1/3 KO mice show no change in CgA-derived peptides ; several peptides derived from CgA were reduced in PC2 KO mouse brains . These investigations demonstrate a novel role for cathepsin L in the production of active catestatin peptide from CgA.
It is of interest that cathepsin L participates in the production of multiple neurotransmitters, as discussed in this section for the production of enkephalin, NPY, β-endorphin, ACTH, α-MSH, CCK, dynorphins, and catestatin. It will be noteworthy in future studies to evaluate the effects of cathepsin L gene knockout mice on behavioral features.
The novel secretory vesicle function of cathepsin L contrasts with its known role in lysosomes for protein degradation. Cathepsin L was first identified as a degrading protease in lysosomes of rat liver and other types of cells and species [48–50]. However, new findings indicate that cathepsin L is present in secretory vesicles of neuroendocrine cells [1–8, 51, 52]. Comparison of the secretory vesicle location of cathepsin L compared to lysosomes reveals differences in the relative portion of cathepsin L in these two organelles in different cell types. In bovine chromaffin cells of the sympathetic nervous system, cathepsin L is primarily colocalized with NPY and enkephalin [2, 3], with essentially no colocalization with the lysosomal marker lamp-1. In pituitary, cathepsin L is present in secretory vesicles with β-endorphin and α-MSH, as well as in lysosomes [4, 53].
Cathepsin L is also present in cell nuclei and extracellularly for biological functions. Nuclear cathepsin L participates in proteolysis of histone H3 in the regulation of gene expression [54, 55]. A truncated form of cathepsin B that lacks the signal peptide is present in the nucleus of several tumor type of cells . Furthermore, a cathepsin L isoform devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor . In addition, extracellular functions of cysteine cathepsins have been demonstrated in matrix degradation and cell signaling and atherosclerosis [58–60].
New findings indicate that cathepsin L is located in several organelles, with notable function in secretory vesicles for neuropeptide production. These recent studies indicate the secretory vesicle as a significant organelle site for cathepsin L production of active peptide neurotransmitters and hormones.
Alzheimer’s disease (AD) is an age-related neurodegenerative disease that results in severe memory deficits. Neurotoxic β-amyloid (Aβ) peptides have been demonstrated as a major factor involved in the development of memory loss of AD, characterized by the accumulation of Aβ in amyloid plaques in AD brains [17–19, 61–63]. Aβ peptides (composed primarily of Aβ(1–40) and Aβ(1–42)) are generated from the amyloid precursor protein (APP) by protease processing by β- and γ-secretase sites located at the N- and C-termini of Aβ within APP. Previously, β- and γ-secretases have been identified as BACE1 and the presenilin complex, respectively [17–19, 61–63]. Recent studies of β-secretase in regulated secretory vesicles show that cathepsin B also participates as β-secretase for Aβ production [9–16], the topic of this section. The AD field has high interest in finding inhibitors of APP processing for reduction of Aβ and improvement in memory deficit.
Investigation of β-secretase in regulated secretory vesicles led to identification of cathepsin B in secretory vesicles as a β-secretase for production of Aβ [9–16]. Inhibition of cathepsin B results in reduction of brain Aβ peptides and significant improvement in memory in a mouse model of AD [13, 15], and cathepsin B gene knockout results in reduced Aβ production in brain . The cathepsin B data combined with that of the previously known BACE1 β-secretase, an aspartyl protease, indicate joint roles of these two proteases for Aβ production . These findings indicate cathepsin B as an exciting new target for development of effective therapeutic agent(s) for AD. The rationale and steps involved in discovery of cathepsin B as a drug target for AD are discussed in this review.
Alzheimer’s disease (AD) results in severe memory loss resulting from neurodegeneration in the brain, caused in large part by accumulation of neurotoxic β-amyloid (Aβ) peptides. The majority of AD patients are afflicted with sporadic AD that is not linked to genetic mutations [64, 65]. A smaller portion of AD patients possess familial forms of AD with inherited genetic mutations, especially mutations of APP and presenilins that result in increased Aβ production and memory deficit when overexpressed in transgenic mouse models of AD [17, 66, 67]. Development of effective therapeutic agents to improve memory in AD is essential for the increasing numbers of AD patients of the growing aged population.
A logical therapeutic approach for AD is to develop drugs which reduce the production of brain Aβ peptides because their accumulation has been demonstrated to be a major factor in causing the disease [17–19, 61–63, 66, 67]. Aβ peptides are produced from the amyloid precursor protein (APP) by proteolytic cleavage at the β-secretase and γ-secretase sites. The majority of AD patients express wild-type (WT) APP of sporadic AD that is not linked to genetic mutations [64, 65], but a large extended family expresses APP containing mutations at these sites  which results in Aβ accumulation and development of the disease [66, 67, 69]. Because the mutant APP produces more Aβ, expression of mutant APP forms has been useful in developing animal models to identify potential AD therapeutics. However, since most AD patients express WT APP, it is essential to investigate drug candidates in models expressing WT APP because different proteases may processing WT compared to mutant APP substrates.
Accumulation of extracellular Aβ results from neuronal production and secretion of Aβ that results in neurodegeneration of neurons in brain regions (hippocampus and cortex) responsible for memory function (Figure 5). Neurons possess the regulated secretory pathway that is utilized for activity-dependent secretion of neurotransmitters, and the constitutive secretory pathway for basal secretion [5, 70–73]. It is important to understand the neurobiology of Aβ production with respect the primary secretory pathway that provides secretion of extracellular Aβ. Evaluation of data of in vivo secretion of Aβ indicates its secretion primarily from the regulated secretory pathway of neurons (summarized in next paragraph). The location of Aβ and its amyloid precursor protein (APP) in regulated secretory vesicles [9, 10, 74–76] indicates that the proteases that convert APP to Aβ are present in the secretory vesicle organelle.
Studies in the field indicate that the majority of extracellular Aβ in brain is provided by activity-dependent secretion from neurons via the regulated secretory pathway. Neurons undergo high frequency, receptor-mediated, electrical activity-dependent secretion of Aβ [77–80]. Neurotransmitters also undergo regulated secretion [5, 70–73]. In parallel to the main regulated secretory pathway for Aβ secretion, a minor portion of Aβ undergoes basal secretion from the constitutive secretory pathway [9, 10]. Studies of peptide neurotransmitter biosynthesis show that different proteases are present in regulated compared to constitutive secretory pathways [5, 73, 81–83]. Therefore, it is important to identify the proteases for β-secretase in the regulated secretory vesicles that provides the majority of secreted, extracellular Aβ.
Studies of neuronal chromaffin cells (in primary culture) from in vivo nervous tissue finds that they naturally produce Aβ in the regulated secretory pathway that provides the majority of secreted Aβ [9, 10]. These regulated secretory vesicles can be purified in high quantity which allows biochemical characterization and purification of proteolytic activity for Aβ production. These secretory vesicles have been used as a model for studies of neurotransmitter synthesizing enzymes that function in brain [5, 84]. These secretory vesicles contain Aβ40 and Aβ42 with full-length APP (with wild-type β-secretase site), indicating the presence of secretases for production of Aβ peptides from APP. Therefore, these model Aβ-producing secretory vesicles were utilized for identification of β-secretase protease(s).
The wild-type β-secretase site of APP is cleaved between Met-↓Asp at the N-terminal side of the Aβ peptide sequence (fig. 6a). The amino acid residues adjacent to the cleavage site are important for recognition of the peptide cleavage site by the protease, according the model of substrate and protease interactions of Schechter and Berger [85, 86]. Cleavage of the wild-type β-secretase site of APP is relevant to the majority of AD patients because they express wild-type APP.
The specificity for peptide substrate cleavage by proteases involves the active site divided into subsites, using the model and nomenclature of Schechter & Berger [85, 86]. This model describes interactions of the protease with the substrate amino acids flanking the cleavage site (figure 6b.i). The amino acids forming the cleaved peptide bond are termed P1-↓P1', with residues at the N-terminal and C-terminal sides of the cleavage site indicated as P1, P2…․ and P1', P2'…․, respectively. The protease active site is viewed as a series of subsites (S1, S2… and S1', S2'…) each accommodating one amino acid residue of the substrate. Protease subsites interact with the polypeptide backbone and with side chains of amino acids of the substrate.
Multiple points of interactions in several subsites are essential for obtaining the high association constants necessary for efficient biological function. Within the active site certain subsites (close or remote from the catalytic site) participate in determining specificity [85, 87–90]. For example, for papain and the cysteine cathepsins , specificity is determined mainly by the hydrophobic S2 subsite that prefers binding to hydrophobic P2 residues. The specificity and activity of caspases, cysteine proteases, is determined by both S1 and S4 remote from the catalytic site . Generally, the P1 to P3 residues are important for protease selectivity for cleavage sites, based on interaction with S1 to S3 subsites of the protease. Therefore, cleavage site-specific assays often incorporate the native P1 to P3 residues.
At the wild-type β-secretase site of APP, the -P3-P2-P1-↓P1’-P2’-P3’- residues are Val-Lys-Met-↓Asp-Ala-Glu- (figure 6b.ii). This wild-type β-secretase site is expressed by the majority of AD patients. However, a mutant β-secretase site is expressed in an extended Swedish family , consisting of mutant Asn-Leu that substitutes for the wild-type P2-P1 residues of Lys-Met. Because the amino acids at the Swedish mutant site differ from the wild-type β-secretase site, proteases that selectively cleave these cleavage sites may exist.
It is desirable to identify the protease(s) cleaving at the wild-type β-secretase site, since it is expressed in >99% of the AD population. Therefore, consideration of P1 to P3 residues that may confer cleavage specificity can be designed as the peptide-MCA substrate Z-Val-Lys-Met-↓MCA representing a substrate mimicking the wild-type β-secretase site.
Processing of the wild-type β-secretase site substrate, Z-Val-Lys-Met-↓MCA, in Aβ-containing secretory vesicles was identified as activity belonging to the cysteine protease family [9, 10]. The wild-type β-secretase activity in Aβ-producing secretory vesicles was labeled by the activity probe DCG-04, a biotinylated form of the cysteine protease inhibitor E64c (fig. 7a). Inhibition by E64c and CA074 (selective inhibitor of cathepsin B) (fig. 7b) , purification, and peptide sequencing of the affinity-labeled protease by mass spectrometry indicated its sequence to be that of cathepsin B (fig. 7c). The colocalization of cathepsin B with Aβ peptides in secretory vesicles was demonstrated by immunoelectron microscopy (fig. 7d) . This finding indicates the secretory vesicle as a new subcellular compartment for cathepsin B function.
Cathepsin B cleavage of the wild-type β-secretase site of the model substrate Z–Val–Lys–Met–↓MCA was compared to the Swedish mutant β-secretase site substrate Z-Val-Asn-Leu-↓MCA (mutant residues are underlined). Cathepsin B shows clear preference for cleaving the WT β-secretase substrate, with essentially no activity for the Swedish mutant β-secretase substrate (Table 3). The distinct preference of cathepsin B for the WT β-secretase site is indicated by its high catalytic efficiency represented by its kcat/Km value of 3.17 × 105 M−1sec1 , indicating an active enzyme.
Analyses with longer peptide substrates also show that cathepsin B cleaves the wild-type β-secretase site [9, 91]. The endogenous cathepsin B purified from Aβ-containing secretory vesicles cleaves the wild-type β-secretase site of the peptide SVKM↓DAEF (arrow shows β-secretase site) . Furthermore, cathepsin B cleaves the internally quenched fluorescent peptide substrates containing the wild-type β-secretase site within RE(Edans)EVKM↓DAEFK(Dabcl)R-NH2 substrate . These long peptide substrates are often used to evaluate protease cleavage of the β-secretase site [9, 91–93].
Aβ is produced in APP- and Aβ-containing secretory vesicles (isolated from neuronal chromaffin cells) after incubation at 37° C . Notably, the specific cathepsin B inhibitor CA074, as well as with the methylated CA074Me form, blocked Aβ production (fig. 8) . Also, E64c, which inhibits cathepsin B and cysteine proteases, blocked Aβ production in the purified secretory vesicles. These data clearly illustrate a role for cathepsin B in Aβ production.
The effect of the cathepsin B inhibitor was also evaluated in neuronal chromaffin cells. CA074Me reduced the amount of Aβ (Aβ40) in regulated secretory vesicles, whose content of Aβ was measured in the culture media after KCl stimulation of the regulated secretory pathway . The CA074Me inhibitor, however, had no effect on the amount of Aβ secreted from the constitutive secretory pathway. These results indicate the cellular role of cathepsin B for production of Aβ in the regulated secretory pathway.
The E64c molecule inhibits cysteine proteases including cathepsin B . CA074 is a selective inhibitor of cathepsin B [94, 95]. The methylated CA074Me form is more cell permeable, and is converted intracellularly by esterases to the selective CA074 inhibitor of cathepsin B [94–96]. Although some evidence shows that CA074Me is less selective than CA074 , cellular esterases convert CA074Me to CA074 . Therefore, the effects of CA074Me are most likely due to the effects of CA074. The experiments with E64c, CA074, and CA074Me together support a role for cathepsin B in Aβ production in regulated secreory vesicles of neuronal-like chromaffin cells.
Inhibitors of cathepsin B improve memory and reduce Aβ in transgenic mice expressing human APP with the wild-type β-secretase site (and the mutation V717 near the γ-secretase site of APP which promotes Aβ production [13. 16]], known as the London APP mouse model of AD . Administration of the CA074Me and E64d inhibitors (via icv route into brain) resulted in substantial improvement in memory deficit in the London AD mice, assessed by the Morris water maze memory test that measures latency time to swim to a hidden platform (Figure 9). Importantly, the reduced latency time after inhibitor treatment indicates substantial improvement in memory, which approached that of normal mice.
Treatment with the CA074Me and E64d inhibitors reduced amyloid plaque and substantially reduced Aβ40 and Aβ42 levels in brain (Figure 10a,b). The CA074Me and E64d inhibitors also reduced brain levels of CTFβ (Figure 10c) derived from APP by β-secretase (Figure 10d). Importantly, mice remained healthy after inhibitor treatment. It will be of interest to conduct pharmacological studies to assess the effective range of inhibitor doses for improving memory and brain Aβ. Most recently, oral administration of E64d resulted in improved memory and reduced brain Aβ with reduce amyloid plaque in AD mice, indicating he feasibility of E64d as a practical oral drug therapeutic for AD . These novel results demonstrate the in vivo effectiveness of cathepsin B inhibitors to improve memory deficit and to reduce in brain Aβ peptides and amyloid plaque load.
Transgenic mice expressing human Swedish mutant APP have been utilized as a mouse model of AD [17, 62, 66, 69]. The Swedish APP possesses the mutant Asn-Leu residues at the β-secretase cleavage that differs from the wild-type sequence of Lys-Met at that site. Most interestingly, administration of the inhibitors of cathepsin B, CA074Me and E64d to Swedish mutant mice (Swedish mutation in the London APP mice, ie., Swe/London APP mice) resulted in no effect on memory deficit in the Swedish mutant APP mice [13, 15]. Furthermore, inhibitors resulted in no change in brain levels of Aβ40 and Aβ42, or CTFβ in mice with the Swedish mutation of APP (Swe/London APP mice).
Notably, knockout of the cathepsin B gene in mice expressing human wild-type APP results in decreased Aβ40 and Aβ42 by 67% in brain (Figure 11), and decreased levels of the C-terminal β-secretase fragment (CTFβ) derived from APP by β-secretase . These data suggest that deletion of the cathepsin B gene decreases β–secretase activity to reduce Aβ production.
In contrast, knockout of cathepsin B in mice expressing human APP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ  The difference in reduction of Aβ in mice expressing human wild-type APP mice, but not in mice expressing human Swedish mutant APP, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express wild-type APP, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in the majority of the AD population.
Other studies have confirmed the lack of effect of cathepsin B knockout on Aβ in mice expressing APP with the Swe mutation . However, the Mueller-Steiner studies did not examine effects of cathepsin B knockout in mice expressing wild-type APP.
Furthermore, gene silencing of cathepsin B by siRNA in normal wild-type hippocampal brain neurons (in primary culture) reduces the amount of Aβ in the regulated secretory pathway . These studies also showed that the CA074Me inhibitor reduces Aβ in the regulated secretory pathway of rat hippocampal neurons, indicating a role for cathepsin B in Aβ production.
Cathepsin B knockout mice appear healthy, showing normal eating, body weight, grooming, and appearance . These results indicate that drug inhibition of cathepsin B will likely have minimal side effects and will be therapeutically safe.
These data from multiple groups validate cathepsin B as a target for development of drug inhibitors to lower Aβ in the majority of AD patients expressing wild-type APP.
There has been much investigation in the field on the aspartyl protease BACE1 that functions as β-secretase [92, 93, 99–104]. While both cathepsin B and BACE1 are present in Aβ secretory vesicles [9, 10], cathepsin B is the primary β-secretase activity in the regulated secretory vesicle that provides the majority of extracellular Aβ. BACE1 has been demonstrated to provide Aβ released from the constitutive secretory pathway, measured in conditioned media from cells [92, 93, 99–104]. Analyses of the BACE1 studies together with that of cathepsin B support the hypothesis for joint roles of these proteases for Aβ production. Thus, the BACE1 results do not preclude cathepsin B as another β-secretase, explained in this section.
The Swe mutant APP was utilized in the field to search for β-secretase, leading to identification of BACE1 [92, 93, 99, 101, 102] which readily cleaves the Swe β-secretase site, with poor efficiency for cleaving the wild-type β-secretase site. BACE1 activity for the wild-type β-secretase substrate is extremely low, illustrated by low kcat/Km values of 40–60 M−1s−1 [102, 105], whereas proteases acting on biological substrates have kcat/Km values that range between ten of thousands to a few millions [85–87, 105]. Cathepsin B, however, has a kcat/Km for cleaving wild-type β-secretase site substrates of 317,000 M−1s−1 [13, 16]. These kinetic properties demonstrate that cathepsin B most effectively cleaves the wild-type β-secretase site of APP for Aβ production.
Since BACE1 cleaves the Swe mutant APP, investigators have utilized transgenic mice expressing human Swe mutant APP as an AD model for studies of BACE1 gene knockout. Indeed, a study with such Swe mutant APP mice  showed that knockout of BACE1 reduced brain Aβ and CTFβ, which provides support for involvement of BACE1 as β-secretase for the Swedish mutant APP. These BACE1 studies, though, do not rule out a role for cathepsin B in production of Aβ from wild-type APP.
Studies of BACE1 knockout mice apparently found that brain homogenates lack brain β-secretase activity . However, because the β-secretase assays contained E64, a potent inhibitor of cathepsin B, the assay could detect the cysteine protease activity of cathepsin B. Therefore, the assays of that study do not rule out cathepsin B as representing β-secretase activity.
A study found lower Aβ levels in media from cultured brain neurons obtained from BACE1 knockout mice which implicated participation of BACE1 . But the Aβ in “conditioned medium” indicates constitutive secretion of Aβ because the neurons were not stimulated to secrete. The study did not measure regulated secretion of Aβ and, therefore, the data do not rule out participation of cathepsin B. The same analysis applies to another study , which also found reduced Aβ in conditioned medium secreted from neurons from BACE1 knockout mice, indicating a role for BACE1 in the constitutive secretory pathway. The study did not address regulated secretion and, therefore, does not rule out cathepsin B participation in Aβ production in the regulated secretory pathway.
Because AD brains possess physiologically normal levels of APP , proteases for APP processing should be effective at normal APP levels. Studies of the effects of BACE1 knockout on Aβ production from normal, endogenous levels of mouse APP showed reduction of brain Aβ1–40 and partial reduction of Aβx-40 ; heterozygous BACE1 gene deletion (BACE1 −/+) has no effect on brain Aβ. Overexpression of BACE1 in wild-type mice with normal levels of APP results in no change in brain Aβ levels, which led that group to conclude that BACE1 “has minimal effect on the level of endogenous Abeta” and that “other factors” must be involved in modulation of Aβ production in adult and aging brain” . The “other factor” may include cathepsin B in Aβ production.
In transgenic mice expressing super high levels of APP, BACE1 gene knockout resulted in decreased brain Aβ levels . These findings indicate that BACE1 can function with very high, non-physiological levels of APP. It is known that protease enzyme activities are dependent on substrate levels, with more activity at higher substrate levels . The important question is what protease target can be inhibited under conditions of normal APP levels to reduce Aβ? This question is addressed by results showing that inhibitors of cathepsin B reduce brain Aβ in brains of normal wild-type guinea pigs [11, 12]. These data indicate that cathepsin B functions at normal levels of APP for Aβ production.
The data in the field indicate joint roles for cathepsin B and BACE1 as β-secretases for Aβ production of AD. Their notable difference is that these proteases differ in their cleavage preferences. Cathepsin B prefers to cleave the wild-type β-secretase site, whereas BACE1 prefers to cleave the Swedish mutant β-secretase site. Since the majority of AD patients express APP with the wild-type β-secretase site, cathepsin B is important to the AD population. Therefore, development of cathepsin B inhibitors may be useful for novel therapeutic strategies in AD.
The functions of cathepsins L and B in secretory vesicles indicate a previously unknown subcellular location for these cathepsins. These cysteine cathepsins, and other cathepsins, have been known to function in lysosomes for protein degradation but recent studies indicate their biological roles in health and disease . The roles of cathepsins L and B for generation of biologically active peptides in secretory vesicles are distinct from their roles in lysosomes. The secretory vesicle environment provides unique conditions for cathepsin functions which include protein systems to maintain conditions of acidic pH, reduction-oxidation regulation, and numerous key functions of the secretory vesicle proteome that are utilized for cathepsins to generate active peptides in health and disease. Therefore, proteomic studies of secretory vesicles have been conducted to reveal the functional protein systems that provide the appropriate environment for cathepsins L and B to generate active peptides.
Proteomic studies of neuropeptide-containing secretory vesicles can identify the functional protein categories in secretory vesicles utilized for neuropeptide production and secretion. Recent examination of proteins in model bovine chromaffin secretory vesicles revealed multiple functional protein categories that participate in secretory vesicle production of neuropeptides and catecholamines for cell-cell communication (Figure 12) [111, 112]. Protein systems involved in vesicular neuropeptide biosynthesis were examined in proteomic studies of soluble and membrane fractions of dense core secretory vesicles purified from chromaffin cells of the sympathetic nervous system. Proteomic results revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Interestingly, these secretory vesicles contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules [113, 114], together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components [115, 116]. These vesicles also contain ATPases that regulate proton translocation , combined with components for signaling and exocytosis of neuropeptides. It will be of interest to compare the proteomics of neuropeptide secretory vesicles with that of synaptic vesicles that secrete classic small molecule neurotransmitters [118–120].
Interestingly, the proteomic data revealed that these secretory vesicles contain proteins involved in neurodegeneration including amyloid precursor protein (APP), huntingtin interacting protein, cystatin 7, ataxin 7, and prion protein . These regulated secretory vesicles may be informative for investigation of how such neurodegenerative diseases may involve secretory vesicle mechanisms.
Overall, knowledge of secretory vesicle proteomes provides novel insights into the biosynthesis and secretion of active peptides utilized for cell-cell communication in neuroendocrine systems, and in Alzheimer’s disease and other human diseases.
Regulation of brain and neurological disease functions utilizes profiles of active peptides that together function to mediate the complex cell-cell communication network among neuroendocrine systems. Changes in physiological functions are represented by alterations in profiles of active peptides in health and disease. Furthermore, proteomic studies will be useful for biomarker applications for monitoring disease status and the effectiveness of therapeutic agents involving peptide regulation of disease functions. Elucidation of the proteomic systems utilized by the secretory vesicle for producing active neuropeptides for health and for generating toxic peptides such as β-amyloid in Alzheimer’s disease and other neurological diseases, can provide insight into new drug targets for novel disease therapeutics.
This review highlights the paradigm-shifting discoveries of the key biological functions of the cysteine cathepsin L for producing active peptides for neurotransmission and of the key role of cathepsin B for production of Aβ involved in the development of Alzheimer’s disease. It is likely that future research will unveil significant roles of cysteine cathepsins in cellular, physiological, and disease conditions. Such knowledge will provide new target strategies for drug discovery to improve health and disease conditions.
The cathepsin L and proteomics research was supported by grants from the National Institutes of Health to V. Hook consisting of R01DA04271, R01NS24553, and R01MH077305 from the NIH. S. Bark was supported by a NIH Mentored Scientist Award (K01DA023065). The authors also appreciate scientific advice by Dr. Shin-Rong Hwang, and technical assistance by Mr. Thomas Toneff, at the Skaggs School of Pharmacy and Pharmaceutical Sciences, Univ. of Calif., San Diego, La Jolla, CA. The authors appreciate collaboration of these studies with Dr. Thomas Reinheckel and Dr. Christoph Peters at the Albert-Ludwigs University, Freiburg, Germany.
The cathepsin B and Alzheimer’s research was supported by the NIA/NIH R21 Grant AG027446 and 1R44AG032784 (to American Life Science Pharmaceuticals (ALSP)), and by an award from the Alzheimer’s Association to V. Hook. V.H. holds equity in ALSP and serves on the Scientific Advisory Board of ALSP. The terms of this agreement have been reviewed by the University of California, San Diego, in accordance with its conflict of interest policies. G.H. holds equity in and is employed by ALSP.
Figures 1–4, ,88–11 of this review are reproduced from their original articles (2, 5, 10, 13, 14) with the kind permission from the Proceeding of the National Academy of Sciences USA, Annual Review of Pharmacology and Toxicology, Biological Chemistry, the Journal of Biology Chemistry, and Biochemical and Biophysical Research Communications.
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