Exploratory studies on CA-15L,an anti-HIV active HIV-1 capsid fragment

Utilizing overlapping fragment peptide libraries covering the whole sequence of an HIV-1 capsid (CA) protein with the addition of an octa-arginyl moiety, we had previously found several peptides with anti-HIV-1 activity. Herein, among these potent CA fragment peptides, CA-15L was examined because this peptide sequence overlaps with Helix 7, a helix region of the CA protein, which may be important for oligomerization of the CA proteins. A CA-15L surrogate with hydrophilic residues, and its derivatives, in which amino acid sequences are shifted toward the C-terminus by one or more residues, were synthesized and their anti-HIV activity was evaluated. In addition, its derivatives with substitution for the Ser149 residue were synthesized and their anti-HIV activity was evaluated because Ser149 might be phosphorylated in the step of degradation of CA protein oligomers. Several active compounds were found and might become new anti-HIV agents and new tools for elucidation of CA functions.     

A cumulative experience examining the effect of natural and synthetic antimicrobial peptides vs. Chlamydia trachomatis.

We tested the activity of 48 structurally diverse antimicrobial peptides against Chlamydia trachomatis, serovar L2. The peptides' activity against C. trachomatis, serovar L2 was measured in 48-h McCoy cell shell vial assays. Peptides of 16-20 amino acids were more active than larger peptides, such as defensins. Beta-sheet protegrins, as well as alpha-helical peptides such as novispirin (G-10) were equally active. Enantiomers were as active as native structures. Moderate-sized circular mini-defensins were less effective against C. trachomatis. Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection.     

Peptide oligomers from ultra-short peptides using sortase

Sortase A catalyzed ligation of ultra-short peptides leads to inter/intra-molecular transpeptidation to form either linear or cyclic oligomers dependent upon the peptide length. Cyclic peptides were the main products for peptides with more than 15aa. However, for ultra-short (<15aa) peptides, cyclic oligomers became predominant in prolonged reactions. Peptides with 1–3 aminoglycines were equally active but peptide oligomers from peptide containing more than one aminoglycine were prone to hydrolysis.     

Stereoselective interactions of peptide inhibitors with the beta-amyloid peptide

Residues 16-20 of the beta-amyloid peptide (A beta) function as a self-recognition element during A beta assembly into fibers. Peptides containing this motif retain the ability to interact with A beta and, in some cases, potently inhibit its assembly. Replacing L- with D-amino acids could stabilize such peptides and permit their evaluation as therapeutic agents for Alzheimer's disease. Here we have assessed the effect that such a chiral reversal has on inhibitory potency. D-enantiomers of five peptides, KLVFFA, KKLVFFA, KFVFFA, KIVFFA, and KVVFFA, were unexpectedly more active as inhibitors in an in vitro fibrillogenesis assay. Circular dichroism showed that D-KLVFFA more effectively prevented A beta adopting the beta-sheet secondary structure correlated with fibrillogenesis. Electron microscopy showed that fiber formation was also more strongly inhibited by D-KLVFFA. Heterochiral inhibition was confirmed using D-A beta, on the principle that enantiomeric proteins exhibit reciprocal chiral biochemical interactions. With D-Abeta, L-KLVFFA was the more potent inhibitor, rather than d-KLVFFA. Most significantly, D-peptides were more potent at reducing the toxicity of both A beta1-40 and A beta 1-42 toward neuronal cells in culture. This unforeseen heterochiral stereoselectivity of A beta for D-peptide inhibitors should be considered during future design of peptide-based inhibitors of A beta neurotoxicity and fibrillogenesis.     

A comparison of the pupariation acceleration activity of pyrokinin-like peptides native to the flesh fly: Which peptide represents the primary pupariation factor?

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L9]Neb-PVK-2, [I9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I9]Neb-PVK-2 proved inactive, whereas [L9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization>cuticular shrinkage>motor program for anterior retraction>motor program for longitudinal contraction congruent to tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.     

Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

Synthetic peptides as model substrates for the study of the specificity of the polycation-stimulated protein phosphatases

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.     

The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2.

Influenza virus matrix protein-derived peptides were synthesized based on the amino acid motifs for HLA-A2 bound self peptides. Among these peptides a nonamer (amino acids 58 through 66: G I L G F V F T L) was found to be 100 to 1000 times more effective than the commonly used peptide 57-68 (K G I L G F V F T L T V) in sensitizing HLA-A2+ target cells to lysis by influenza virus specific cytotoxic T lymphocytes. The sensitizing activity of the 12-mer 57-68 was not due to contamination with shorter and more active peptides. Intracellular expression of peptide 58-66 (mediated by a stable expression plasmid with DNA coding for this peptide) also sensitized HLA-A2+ cells to lysis. Peptide 58-66 stimulated human PBMC to generate CTL that recognized peptides 58-66 and 57-68 in association with HLA-A2.     

Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH

The Thermotoga neapolitana xylose isomerase (TNXI) is extremely thermostable and optimally active at 95 degrees C. Its derivative, TNXI Val185Thr (V185T), is the most active type II xylose isomerase reported, with a catalytic efficiency of 25.1 s(-1) mM(-1) toward glucose at 80 degrees C (pH 7.0). To further optimize TNXI's potential industrial utility, two rounds of random mutagenesis and low temperature/low pH activity screening were performed using the TNXI V185T-encoding gene as the template. Two highly active mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S). 1F1 was more active than 3A2, which in turn was more active than TNXI V185T at all temperatures and pH values tested. 3A2 and 1F1's high activities at low temperatures were due to significantly lower activation energies (57 and 44 kJ/mol, respectively) than that of TNXI and V185T (87 kJ/mol). Mutation L282P introduced a kink in helix alpha7 of 3A2's (alpha/beta)8 barrel. Surprisingly, this mutation kinetically destabilized 3A2 only at pH 5.5. 1F1 displayed kinetic stability slightly above that of TNXI V185T. In 1F1, mutation F186S creates a cavity that disrupts a four-residue network of aromatic interactions. How the conformation of the neighboring residues is affected by this cavity and how these conformational changes increase 1F1's stability still remain unclear.     

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.     

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis the solution structure of aurein 1.2.

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.     

Potent cytolytic response by a CD8+ CTL clone to multiple peptides from the same protein in association with an allogeneic class I MHC molecule

CTL clone 2C recognizes the allogeneic class I MHC molecule L(d) in association with peptides derived from alpha-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L(d) than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L(d) as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.     

Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides.

Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C alpha subunit. Ki values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher Km for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (1991) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.     

Enhanced antitumor activity and selectivity of lactoferrin-derived peptides.

A number of peptide analogs derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT-29 and MT-1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure-activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.     

Analysis of the two-peptide bacteriocins lactococcin G and enterocin 1071 by site-directed mutagenesis

The two peptides (Lcn-alpha and Lcn-beta) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-alpha and Ent-beta) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-alpha+Ent-beta had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-alpha+Lcn-beta), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-alpha+Lcn-beta) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-alpha+Ent-beta), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-alpha is more active against lactococci in combination with Lcn-beta and more active against enterococci in combination with Ent-beta suggests that the beta peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the beta peptide seem to be important for specificity, since Ent-alpha combined with an Ent-beta variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-alpha+Ent-beta. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-beta had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the alpha peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-alpha influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2- to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only approximately 2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-alpha and Lcn-beta, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an approximately 10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-alpha and Lcn-beta, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in an Lcn-Ent-beta hybrid peptide was more detrimental when the altered peptide was combined with Lcn-alpha (>10-fold reduction) than when it was combined with Ent-alpha ( approximately 2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif) in the beta peptide may be involved in a specific interaction with the cognate alpha peptide. It is also noteworthy that the K2P and A7P mutations in Lcn-beta reduced the activity only approximately 2-fold, suggesting that the first seven residues in the beta peptides do not form an alpha-helix.     

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.