Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the beta-propeller and alpha/beta-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln [downward arrow]Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. PstI was chosen as a 5' cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL(pro) (T25G) was found to have a 3-fold improvement over TEV(pro) in tag cleavage at each respective preferred cleavage site.

The pattern-recognition receptor NOD2 senses bacterial muropeptides to regulate host immunity and maintain homeostasis. Loss-of-function mutations in NOD2 are associated with Crohn's disease (CD), but how the variations in microbial factors influence NOD2 signaling and host pathology is elusive. We demonstrate that the Firmicutes peptidoglycan remodeling enzyme, DL-endopeptidase, increased the NOD2 ligand level in the gut and impacted colitis outcomes. Metagenomic analyses of global cohorts (n = 857) revealed that DL-endopeptidase gene abundance decreased globally in CD patients and negatively correlated with colitis. Fecal microbiota from CD patients with low DL-endopeptidase activity predisposed mice to colitis. Administering DL-endopeptidase, but not an active site mutant, alleviated colitis via the NOD2 pathway. Therapeutically restoring NOD2 ligands with a DL-endopeptidase-producing Lactobacillus salivarius strain or mifamurtide, a clinical analog of muramyl dipeptide, exerted potent anti-colitis effects. Our study suggests that the depletion of DL-endopeptidase contributes to CD pathogenesis through NOD2 signaling, providing a therapeutically modifiable target.

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder characterized by high levels of cholestanol in the blood and accumulation of cholestanol in multiple tissues, especially the brain, often presents in parkinsonism. However, it remains unknown whether cholestanol plays a role in the pathogenesis of sporadic Parkinson's disease (PD). Here, we show that the levels of serum cholestanol in patients with sporadic PD are higher than those in control participants. Cholestanol activates the protease asparagine endopeptidase (AEP) and induces the fragmentation of α-synuclein (α-syn) and facilitates its aggregation. Furthermore, cholestanol promotes the spreading of α-syn pathology in a mouse model induced by intrastriatal injection of α-syn fibrils. KO of AEP or administration of an AEP inhibitor ameliorates α-syn pathology, degeneration of the nigrostriatal dopaminergic pathway, and PD-like motor symptoms. These results not only indicate that cholestanol contributes to the aggregation and spreading of α-syn by activating AEP but also reveal an opportunity for treating PD with AEP inhibitors.

Natriuretic peptides are cyclic vasoactive peptide hormones with great diagnostic and therapeutic relevance. The main catabolic pathway postulated for natriuretic peptides is the degradation by neutral endopeptidase (NEP). However, B-type natriuretic peptide has been found to be resistant to NEP. Here, we compared the degradation of various mature, truncated, and recombinant natriuretic peptides by NEP. The degradation was clearly dependent on the length of the N- or C-terminus as well as on distinct sequence differences within the essential loop structure of the natriuretic peptides. Based on these findings, we developed a model for the interaction of NEP and natriuretic peptides that enables new insights into the mode of action and prediction of substrates of NEP, a peptidase that plays a key role in crucial (patho-) physiological processes.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway. Synaptic dysfunction impairs dopamine turnover and contributes to the degeneration of dopaminergic neurons. However, the molecular mechanisms underlying synaptic dysfunction and dopaminergic neuronal vulnerability in PD are not clear. Here, we report that synaptojanin 1 (SYNJ1), a polyphosphoinositide phosphatase concentrated at nerve terminals, is a substrate of a cysteine proteinase, asparagine endopeptidase (AEP). SYNJ1 is cleaved by the cysteine proteinase AEP at N599 in the brains of PD patients. AEP-mediated cleavage of SYNJ1 disrupts neuronal phosphoinositide homeostasis and causes synaptic dysfunction. Overexpression of the AEP-generated fragments of SYNJ1 triggers synaptic dysfunction and the degeneration of dopaminergic neurons, inducing motor defects in the α-synuclein transgenic mice. Blockage of AEP-mediated cleavage of SYJN1 alleviates the pathological and behavioral defects in a mouse model of PD. Our results demonstrate that the fragmentation of SYNJ1 by AEP mediates synaptic dysfunction and dopaminergic neuronal degeneration in PD.

The impermeability barrier provided by the outer membrane of enteric bacteria, a feature lacking in Gram-positive bacteria, plays a major role in maintaining resistance to numerous antimicrobial compounds and antibiotics. Here we demonstrate that mutational inactivation of spr, coding for a muramyl endopeptidase, significantly sensitizes Salmonella enterica serovar Typhimurium to vancomycin without any accompanying apparent growth defect or outer membrane destabilization. A similar phenotype was not achieved by deleting the genes coding for muramyl endopeptidases MepA, PbpG, NlpC, YedA, or YhdO. The spr mutant showed increased autolytic behavior in response to not only vancomycin, but also to penicillin G, an antibiotic for which the mutant displayed a wild-type MIC. A screen for suppressor mutations of the spr mutant phenotype revealed that deletion of tsp (prc), encoding a periplasmic carboxypeptidase involved in processing Spr and PBP3, restored intrinsic resistance to vancomycin and reversed the autolytic phenotype of the spr mutant. Our data suggest that Spr contributes to intrinsic antibiotic resistance in S. Typhimurium without directly affecting the outer membrane permeability barrier. Furthermore, our data suggests that compounds targeting specific cell wall endopeptidases might have the potential to expand the activity spectrum of traditional Gram-positive antibiotics.

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S-transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S-transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

Neurofibrillary tangles composed of hyperphosphorylated tau and synaptic dysfunction are characteristics of Alzheimer's disease (AD). However, the underlying molecular mechanisms remain poorly understood. Here, we identified Amphiphysin I mediates both tau phosphorylation and synaptic dysfunction in AD. Amphiphysin I is cleaved by a cysteine proteinase asparagine endopeptidase (AEP) at N278 in the brains of AD patients. The amount of AEP-generated N-terminal fragment of Amphiphysin I (1-278) is increased with aging. Amphiphysin I (1-278) inhibits clathrin-mediated endocytosis and induces synaptic dysfunction. Furthermore, Amphiphysin I (1-278) binds p35 and promotes its transition to p25, thus activates CDK5 and enhances tau hyperphosphorylation. Overexpression of Amphiphysin I (1-278) in the hippocampus of Tau P301S mice induces synaptic dysfunction, tau hyperphosphorylation, and cognitive deficits. However, overexpression of the N278A mutant Amphiphysin I, which resists the AEP-mediated cleavage, alleviates the pathological and behavioral defects. These findings suggest a mechanism of tau hyperphosphorylation and synaptic dysfunction in AD.

Accumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties.

Secondary activation of the endothelin system is thought to be involved in toxic liver injury. This study tested the hypothesis that dual endothelin-converting enzyme / neutral endopeptidase blockade might be able to attenuate acute toxic liver injury. - Male Sprague-Dawley rats were implanted with subcutaneous minipumps to deliver the novel compound SLV338 (10 mg/kg*d) or vehicle. Four days later they received two intraperitoneal injections of D-galactosamine (1.3 g/kg each) or vehicle at an interval of 12 hours. The animals were sacrificed 48 hours after the first injection. - Injection of D-galactosamine resulted in very severe liver injury, reflected by strongly elevated plasma liver enzymes, hepatic necrosis and inflammation, and a mortality rate of 42.9 %. SLV338 treatment did not show any significant effect on the extent of acute liver injury as judged from plasma parameters, hepatic histology and mortality. Plasma measurements of SLV338 confirmed adequate drug delivery. Plasma concentrations of big endothelin-1 and endothelin-1 were significantly elevated in animals with liver injury (5-fold and 62-fold, respectively). Plasma endothelin-1 was significantly correlated with several markers of liver injury. SLV338 completely prevented the rise of plasma big endothelin-1 (p<0.05) and markedly attenuated the rise of endothelin-1 (p = 0.055). - In conclusion, dual endothelin-converting enzyme / neutral endopeptidase blockade by SLV338 did not significantly attenuate D-galactosamine-induced acute liver injury, although it largely prevented the activation of the endothelin system. An evaluation of SLV338 in a less severe model of liver injury would be of interest, since very severe intoxication might not be relevantly amenable to pharmacological interventions.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.

Discovery of early-stage biomarkers is a long-sought goal of Alzheimer's disease (AD) diagnosis. Age is the greatest risk factor for most AD and accumulating evidence suggests that age-dependent elevation of asparaginyl endopeptidase (AEP) in the brain may represent a new biological marker for predicting AD. However, this speculation remains to be explored with an appropriate assay method because mammalian AEP exists in many organs and the level of AEP in body fluid isn't proportional to its concentration in brain parenchyma. To this end, we here modified gold nanoparticle (AuNPs) into an AEP-responsive imaging probe and choose transgenic APPswe/PS1dE9 (APP/PS1) mice as an animal model of AD. Our aim is to determine whether imaging of brain AEP can be used to predict AD pathology.

Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM-1 s-1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) are expressed in human osteoblasts and mediate fracture healing. BDNF/TrkB signaling activates Akt that phosphorylates and inhibits asparagine endopeptidase (AEP), which regulates the differentiation fate of human bone marrow stromal cells (hBMSC) and is altered in postmenopausal osteoporosis. Here we show that R13, a small molecular TrkB receptor agonist prodrug, inhibits AEP and promotes bone formation. Though both receptor activator of nuclear factor kappa-Β ligand (RANK-L) and osteoprotegerin (OPG) induced by ovariectomy (OVX) remain comparable between WT and BDNF+/- mice, R13 treatment significantly elevates OPG in both mice without altering RANKL, blocking trabecular bone loss. Strikingly, both R13 and anti-RANK-L exhibit equivalent therapeutic efficacy. Moreover, OVX increases RANK-L and OPG in WT and AEP KO mice with RANK-L/OPG ratio lower in the latter than the former, attenuating bone turnover. 7,8-DHF, released from R13, activates TrkB and its downstream effector CREB, which is critical for OPG augmentation. Consequently, 7,8-DHF represses C/EBPβ/AEP pathway, inhibiting RANK-L-induced RAW264.7 osteoclastogenesis. Therefore, our findings support that R13 exerts its therapeutic efficacy toward osteoporosis via inhibiting AEP and escalating OPG.