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

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.     

Unique neuronal functions of cathepsin L and cathepsin B in secretory vesicles: biosynthesis of peptides in neurotransmission and neurodegenerative disease

Proteases are required for the production of peptide neurotransmitters and toxic peptides in neurodegenerative diseases. Unique roles of the cysteine proteases cathepsin L and cathepsin B in secretory vesicles for the production of biologically active peptides have been demonstrated in recent studies. Secretory vesicle cathepsin L participates in the proteolytic conversion of proenkephalin into the active enkephalin, an opioid peptide neurotransmitter that mediates pain relief. Moreover, recent findings provide evidence that cathepsin B in regulated secretory vesicles participates in the production of toxic beta-amyloid peptides that are known to accumulate extracellularly in Alzheimer's disease brains. The neurobiological functions of cathepsins L and B demonstrate that these secretory vesicle cysteine proteases produce biologically active peptides. These results demonstrate newly identified roles for cathepsins L and B in neurosecretory vesicles in the production of biologically active peptides.     

Protease Pathways in Peptide Neurotransmission and Neurodegenerative Diseases

1. Recent research demonstrates the critical importance of neuroproteases for the production of peptide neurotransmitters, and for the production of toxic peptides in major neurodegenerative diseases that include Alzheimer's (AD) and Huntington's diseases. This review describes the strategies utilized to identify the appropriate proteases responsible for producing active peptides for neurotransmission, with application of such approaches for defining protease mechanisms in neurodegenerative diseases.2. Integration of multidisciplinary approaches in neurobiology, biochemistry, chemistry, proteomics, molecular biology, and genetics has been utilized for neuroprotease studies. These investigations have identified secretory vesicle cathepsin L for the production of the enkephalin opioid peptide neurotransmitter and other neuropeptides. Furthermore, new results using these strategies have identified secretory vesicle cathepsin B for the production of β-amyloid (Aβ) in the major regulated secretory pathway that provides activity-dependent secretion of Aβ peptides, which accumulate in AD.3. CNS neuroproteases that participate in peptide neurotransmission and in neurodegenerative diseases represent new candidate drug targets that may be explored in future research for the development of novel therapeutic agents for neurological conditions.     

Cathepsin B, L, and S cleave and inactivate secretory leucoprotease inhibitor.

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Proteomics of neuroendocrine secretory vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.     

Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.     

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore , in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.     

Cysteine cathepsins: cellular roadmap to different functions

Cysteine cathepsins belong to the papain-like family C1 of clan CA cysteine peptidases. These enzymes are ubiquitously expressed and exert their proteolytic activity mainly, but not exclusively within the compartments along the endocytic pathway. Moreover, cysteine cathepsins are active in pericellular environments as soluble enzymes or bound to cell surface receptors at the plasma membrane, and possibly even within secretory vesicles, the cytosol, mitochondria, and within the nuclei of eukaryotic cells. Proteolytic actions performed by cysteine cathepsins are essential in the maintenance of homeostasis and depend heavily upon their correct sorting and trafficking within cells. As a consequence, the numerous and diverse approaches to identification, qualitative and quantitative determination, and visualization of cysteine cathepsin functions in vitro, in situ, and in vivo cover the entire spectrum of biochemistry, molecular and cell biology. This review focuses upon the transport pathways directing cysteine cathepsins to their points of action and thus emphasizes the broader role and functionality of cysteine cathepsins in a number of specific cellular locales. Such understanding will provide a foundation for future research investigating the involvement of these peptidases with their substrates, inhibitors, and the intertwined proteolytic networks at the hubs of complex biological systems.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.     

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L.

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cysteine proteases, which colocalize with their circulating, tight-binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cathepsins B, K and L to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens. Although both cathepsins processed high-molecular weight kininogen under stoichiometric conditions, only cathepsin L generated significant amounts of immunoreactive kinins. Cathepsin L exhibited higher specificity constants (kcat/Km) than tissue kallikrein (hK1), and similar Michaelis constants towards kininogen-derived synthetic substrates. A 20-mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys-bradykinin (20%) when incubated with cathepsin L. By contrast, cathepsin K did not release any kinin, but a truncated kinin metabolite BK(5-9) [FSPFR(385-389)]. Accordingly cathepsin K rapidly produced BK(5-9) from bradykinin and Lys-bradykinin, and BK(5-8) from des-Arg9-bradykinin, by cleaving the Gly384-Phe385 bond. Data suggest that extracellular cysteine proteases may participate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase.     

Cysteine Cathepsins Activate ELR Chemokines and Inactivate Non-ELR Chemokines

Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9–12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca²⁺ mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9–12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation.     

Cathepsin H functions as an aminopeptidase in secretory vesicles for production of enkephalin and galanin peptide neurotransmitters

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

3-Acylamino-azetidin-2-one as a novel class of cysteine proteases inhibitors

A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the beta-lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar IC(50) values.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.