Search

Protein modification by enzymatic breaking and forming of peptide bonds significantly expands the repertoire of genetically encoded protein sequences. The dual protease-ligase legumain exerts the two opposing activities within a single protein scaffold. Primarily localized to the endolysosomal system, legumain represents a key enzyme in the generation of antigenic peptides for subsequent presentation on the MHCII complex. Here we show that human legumain catalyzes the ligation and cyclization of linear peptides at near-neutral pH conditions, where legumain is intrinsically unstable. Conformational stabilization significantly enhanced legumain’s ligase activity, which further benefited from engineering the prime substrate recognition sites for improved affinity. Additionally, we provide evidence that specific legumain activation states allow for differential regulation of its activities. Together these results set the basis for engineering legumain proteases and ligases with applications in biotechnology and drug development.

Brain barrier is both a protective permeability hurdle and a limitation site where therapeutic agents are excluded to enter the target region. Designing drug vehicle to overcome this notorious barrier bottle is challenging. Herein, we construct a stimuli-responsive self-assembled nanovesicle that delivers water-soluble drugs to prevent the efflux transport of brain barriers by responding to the endogenously occurring signals in Alzheimer's disease (AD) brain microenvironment. Once stimuli-responsive vesicles are accumulated in intracerebrally, the intrinsically occurring legumain endopeptidase cleaves the Ac-Ala-Ala-Asn-Cys-Asp (AK) short peptide on the drug vesicles to expose the 1,2 thiol amino group to cyclize with the cyano groups on 2-cyano-6-aminobenzothiazole (CABT) of the chaperone vesicles, thus triggering the formation of cross-linked micrometre-scale vesicles. Such a structural alteration completely prevents further brain barriers efflux. The superior neuroprotective capacity of cross-linked vesicles is validated in senescence accelerated mouse prone 8 (SAMP8). This smart multi-drug delivery vesicle is promising to serve as a powerful system for AD treatment and can be adapted for the therapy of other central nervous system (CNS) disorders., Graphical abstract a) Responsive cross-linked of large compound vesicles formed in intracerebrally. b) Inhibition of brain barrier efflux and synergistic treatment of AD.Image 1, Highlights • Drug efflux is a bottleneck for treating many disorders, such as Alzheimer's diseases (AD). • The key to success in drug brain parenchyma anchoring is specific targeting delivery. • Functionalized vesicles provided the possibility of inhibiting the efflux transport and increasing drug brain enrichment. • Legumain-induced aggregated multi-drug delivery vesicle is promising to serve as a powerful system for AD treatment.

Background Currently, there is no cure for Alzheimer’s disease (AD). Therapeutics that can modify the early stage of AD are urgently needed. Recent studies have shown that the pathogenesis of AD is closely regulated by an endo/lysosomal asparaginyl endopeptidase (AEP). Inhibition of AEP has been reported to prevent neural degeneration in transgenic mouse models of AD. However, more than 90% of AD cases are age-related sporadic AD rather than hereditary AD. The therapeutic efficacy of AEP inhibition in ageing-associated sporadic AD remains unknown. Methods The senescence-accelerated mouse prone 8 (SAMP8) was chosen as an approximate model of sporadic AD and treated with a selective AEP inhibitor,: δ-secretase inhibitor 11. Activation of AEP was determined by enzymatic activity assay. Concentration of soluble amyloid β (Aβ) in the brain was determined by ELISA. Morris water maze test was performed to assess the learning and memory-related cognitive ability. Pathological changes in the brain were explored by morphological and western blot analyses. Results The enzymatic activity of AEP in the SAMP8 mouse brain was significantly higher than that in the age-matched SAMR1 mice. The half maximal inhibitory concentration (IC50) for δ-secretase inhibitor 11 to inhibit AEP in vitro is was around 150 nM. Chronic treatment with δ-secretase inhibitor 11 markedly decreased the brain AEP activity, reduced the generation of Aβ1–40/42 and ameliorated memory loss. The inhibition of AEP with this reagent not only reduced the AEP-cleaved tau fragments and tau hyperphosphorylation, but also attenuated neuroinflammation in the form of microglial activation. Moreover, treatment with δ-secretase inhibitor 11 prevented the synaptic loss and alleviated dendritic disruption in SAMP8 mouse brain. Conclusions Pharmacological inhibition of AEP can intervene and prevent AD-like pathological progress in the model of sporadic AD. The up-regulated AEP in the brain could be a promising target for early treatment of AD. The δ-secretase inhibitor 11 can be used as a lead compound for translational development of AD treatment.

Exploitation of stimuli-responsive nanoplatforms is of great value for precise and efficient cancer theranostics. Herein, an in situ activable “nanocluster-bomb” detonated by endogenous overexpressing legumain is fabricated for contrast-enhanced tumor imaging and controlled gene/drug release. By utilizing the functional peptides as bioligands, TAMRA-encircled gold nanoclusters (AuNCs) endowed with targeting, positively charged and legumain-specific domains are prepared as quenched building blocks due to the AuNCs' nanosurface energy transfer (NSET) effect on TAMRA. Importantly, the AuNCs can shelter therapeutic cargos of DNAzyme and Dox (Dzs-Dox) to aggregate larger nanoparticles as a “nanocluster-bomb” (AuNCs/Dzs-Dox), which could be selectively internalized into cancer cells by integrin-mediated endocytosis and in turn locally hydrolyzed in the lysosome with the aid of legumain. A “bomb-like” behavior including “spark-like” appearance (fluorescence on) derived from the diminished NSET effect of AuNCs and cargo release (disaggregation) of Dzs-Dox is subsequently monitored. The results showed that the AuNC-based disaggregation manner of the “nanobomb” triggered by legumain significantly improved the imaging contrast due to the activable mechanism and the enhanced cellular uptake of AuNCs. Meanwhile, the in vitro cytotoxicity tests revealed that the detonation strategy based on AuNCs/Dzs-Dox readily achieved efficient gene/chemo combination therapy. Moreover, the super efficacy of combinational therapy was further demonstrated by treating a xenografted MDA-MB-231 tumor model in vivo. We envision that our multipronged design of theranostic “nanocluster-bomb” with endogenous stimuli-responsiveness provides a novel strategy and great promise in the application of high contrast imaging and on-demand drug delivery for precise cancer theranostics., An in situ activable “nanocluster-bomb” detonated by endogenous overexpressing legumain is fabricated for contrast enhanced cancer imaging and effective gene/chemo-therapy.

Legumain is required for maintenance of normal kidney homeostasis. However, its role in acute kidney injury (AKI) is still unclear. Here, we induced AKI by bilateral ischemia-reperfusion injury (IRI) of renal arteries or folic acid in lgmnWT and lgmnKO mice. We assessed serum creatinine, blood urea nitrogen, histological indexes of tubular injury, and expression of KIM-1 and NGAL. Inflammatory infiltration was evaluated by immunohistological staining of CD3 and F4/80, and expression of TNF-α, CCL-2, IL-33, and IL-1α. Ferroptosis was evaluated by Acsl4, Cox-2, reactive oxygen species (ROS) indexes H2DCFDA and DHE, MDA and glutathione peroxidase 4 (GPX4). We induced ferroptosis by hypoxia or erastin in primary mouse renal tubular epithelial cells (mRTECs). Cellular survival, Acsl4, Cox-2, LDH release, ROS, and MDA levels were measured. We analyzed the degradation of GPX4 through inhibition of proteasomes or autophagy. Lysosomal GPX4 was assessed to determine GPX4 degradation pathway. Immunoprecipitation (IP) was used to determine the interactions between legumain, GPX4, HSC70, and HSP90. For tentative treatment, RR-11a was administrated intraperitoneally to a mouse model of IRI-induced AKI. Our results showed that legumain deficiency attenuated acute tubular injury, inflammation, and ferroptosis in either IRI or folic acid-induced AKI model. Ferroptosis induced by hypoxia or erastin was dampened in lgmnKO mRTECs compared with lgmnWT control. Deficiency of legumain prevented chaperone-mediated autophagy of GPX4. Results of IP suggested interactions between legumain, HSC70, HSP90, and GPX4. Administration of RR-11a ameliorated ferroptosis and renal injury in the AKI model. Together, our data indicate that legumain promotes chaperone-mediated autophagy of GPX4 therefore facilitates tubular ferroptosis in AKI.

Neuroinflammation is the important pathological feature of Alzheimer's disease (AD). Legumain, a lysosomal cysteine protease, plays an important role in neuroinflammation during ischemic stroke and depressive disorder. Legumain is involved in AD process through cleaving APP; however, it is unclear if legumain can possibly modulate neuroinflammation without cleaving APP in AD. Thus, we established a mouse model of AD by single intracerebroventricular injections of Aβ1-42 in legumain knockout (KO) mice. The behavioral tests showed that legumain-KO effectively ameliorated cognitive impairment induced by Aβ1-42. Moreover, legumain deprivation significantly improves the synaptic plasticity damages in Aβ1-42-treated mice. Moreover, legumain-KO considerably inhibited the activation of microglia and reduced the expression of inflammatory cytokines in the hippocampus of Aβ1-42-treated mice. Interestingly, we found that legumain-KO inhibited TLR4/MyD88/NF-κB pathway, which was activated by Aβ1-42 in the hippocampus. In conclusion, our results suggested that legumain-KO reduced the level of neuroinflammation that was associated with inhibiting TLR4/MyD88/NF-κB pathways, thereby improving the hippocampal synaptic plasticity and reducing the cognitive impairments in Aβ1-42-treated mice. Legumain knockout blocked microglia activation by inhibiting TLR4/MyD88/NF-κB signaling pathways, and further reduced inflammatory cytokine expression. As a result, legumain knockout alleviated synaptic damage and cognitive impairment induced by Aβ1--42.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1.Par2Nav1.8andLgmndeletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice.Par2deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2by biased mechanisms in HEK293 cells to induce Ca2+mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR2endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENTOral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2. Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2. Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR2endocytosis. Thus, Lgmn is a biased agonist of PAR2that evokes cancer pain.

Several antibody-drug conjugates (ADCs) have failed to achieve a sufficiently large therapeutic window in patients due to toxicity induced by unspecific payload release in the circulation or ADC uptake into healthy organs. Herein, we describe the successful engineering of ADCs consisting of novel linkers, which are efficiently and selectively cleaved by the tumor-associated protease legumain. ADCs generated via this approach demonstrate high potency and a preferential activation in tumors compared to healthy tissue, thus providing an additional level of safety. A remarkable tolerance of legumain for different linker peptides, including those with just a single asparagine residue, together with a modifier of the physicochemical metabolite profile, proves the broad applicability of this approach for a tailored design of ADCs.

Herein we first report a dual-responsive peptide substrate (Comp. 1) for preparing self-assembled nanomaterials triggered by pH and legumain. The dual-responsive self-assembly of Comp. 1 in glioma cells enables its long retention time in lysosomes, S phase arrest, and cell growth locking. We verified that the blocked degradation of HIF-1α in lysosomes played a key role in cell cycle arrest and decreased DNA replication. This work illustrates the disturbance of lysosomal function by self-assembled nanomaterials as a promising strategy for inhibiting glioma cell growth.

Tumor-associated macrophages (TAMs) play a crucial role in the tumor microenvironment. Legumain (LGMN) has been shown to be a tumor-promoting protein, but the effect of LGMN on TAMs in the progression of gastric cancer (GC) is under exploration. Our studies included the construction of LGMN-knockdown and LGMN-overexpressing TAMs induced from the human cell line THP-1 (PMA/IL-4/IL-13) and murine cell line Raw264.7 (IL-4/IL-13). A CCK-8 assay and transwell migration assay indicated that upregulation of LGMN expression in TAMs stimulated cell proliferation, migration and invasion in vitro, while downregulation of LGMN expression reduced cell proliferation, migration and invasion. In vivo experiments revealed slower growth, less angiogenesis, and less Ki67 expression in LGMN-knockdown TAMs injected with gastric cancer cells compared to control TAMs injected with GC cells. Together, these study results suggested that LGMN+ TAMs, which may serve as a potential target for GC treatment, promoted gastric cancer cell proliferation and angiogenesis in vitro and in vivo.

Overexpression of legumain is closely associated with tumor proliferation, invasion, and metastasis. Because of its intrinsic properties, such as high sensitivity and resolution, positron emission tomography (PET) has become an effective imaging technique for early diagnosis, treatment response prediction, and monitoring. Herein, two legumain-targeting radiofluorinated smart probes (18F-2 and 18F-3) as well as a control probe (18F-1) were specifically designed for PET imaging of legumain activity in tumors. 18F-1, 18F-2, and 18F-3 were obtained with high radiochemical yield (RCY > 60%) and radiochemical purity (RCP > 99%) using a convenient "one-step" 18F-labeling method. The probes 18F-2 and 18F-3 exhibited high response to legumain activity and reductive environment and revealed comparable uptake in HCT116 cells (4.22% ± 0.14% and 4.64% ± 0.32% for 18F-2 and 18F-3, respectively; 8.46% ± 0.33% and 9.05% ± 0.24% for co-treatment of 18F-2 + 2 and 18F-3 + 3 at 1 h), while the control probe 18F-1 showed no response. PET imaging of tumor-bearing mice showed that the co-injection strategy (18F-2 + 2 and 18F-3 + 3) resulted in higher tumor uptake (3.57% ± 0.37% and 3.72% ± 0.19% ID/g at 10 min, respectively) than the single injection strategy (2.59% ± 0.19% and 2.60% ± 0.46% ID/g for 18F-2 and 18F-3, respectively). In addition, introduction of the trimeric histidine-glutamate (HEHEHE) tag to 18F-3 reduced the liver uptake by almost two-fold without any noticeable effect on the tumor uptake. All the results indicate that 18F-3 holds great potential applications in clinics for sensitive and specific PET imaging of legumain activity in tumors.

Doxorubicin (DOX) is widely used as a chemotherapeutic agent for liver cancer. However, its clinical applications are greatly restricted by its nonselective cytotoxicity. A novel magnetic prodrug, Fe3O4@DOX, was designed, synthesized and characterized, and Fe3O4 and DOX were connected by the peptide CGGAAN. The magnetic prodrug Fe3O4@DOX was successfully synthesized with average sizes of 95 nm and 322.5 nm by TEM (transmission electron microscopy) and Malvern Zetasizer instrument respectively. The maximum emission wavelength shifted from 594 nm for free DOX to 615 nm for conjugated DOX in the synthesized Fe3O4@DOX. Both free DOX and Fe3O4@DOX show strong cytotoxicity to legumain overexpressing PLC through apoptosis. Similarly, Fe3O4@DOX and DOX equally reduced tumor volume and induced cell apoptosis in tumor tissues, while the former significantly maintained body weight and extended the life of nude mice, therefore serving as a promising nanocarrier for liver cancer treatment.

Summary Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux and reduced proliferation of CD4 T-cells. In antigen presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease - a prototypic CD4 T-cell-mediated disease - and found that HCQ limits ex vivo antigen-specific T cell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.

Legumain (asparaginyl endopeptidase) is the only protease with a preference for cleavage after asparagine residues. Increased legumain activity is a hallmark of inflammation, neurodegenerative diseases, and cancer, and legumain inhibitors have exhibited therapeutic effects in mouse models of these pathologies. Improved knowledge of its substrates and cellular functions is a requisite to further validation of legumain as a drug target. We, therefore, aimed to investigate the effects of legumain inhibition in macrophages using an unbiased and systematic approach. By shotgun proteomics, we identified 16 094 unique peptides in RAW264.7 cells. Among these, 326 unique peptides were upregulated in response to legumain inhibition, while 241 were downregulated. Many of these proteins were associated with mitochondria and metabolism, especially iron metabolism, indicating that legumain may have a previously unknown impact on related processes. Furthermore, we used N-terminomics/TAILS (terminal amine isotopic labeling of substrates) to identify potential substrates of legumain. We identified three new proteins that are cleaved after asparagine residues, which may reflect legumain-dependent cleavage. We confirmed that frataxin, a mitochondrial protein associated with the formation of iron-sulfur clusters, can be cleaved by legumain. This further asserts a potential contribution of legumain to mitochondrial function and iron metabolism. Lastly, we also identified a potential new cleavage site within legumain itself that may give rise to a 25 kDa form of legumain that has previously been observed in multiple cell and tissue types. Collectively, these data shed new light on the potential functions of legumain and will be critical for understanding its contribution to disease.

Glioma treatment using targeted chemotherapy is still far from satisfactory due to not only the limited accumulation but also the multiple survival mechanisms of glioma cells, including up-regulation of both autophagy and programmed cell death ligand 1 (PD-L1) expression. Herein, we proposed a combined therapeutic regimen based on functional gold nanoparticles (AuNPs)-enabled chemotherapy, autophagy inhibition, and blockade of PD-L1 immune checkpoint. Specifically, the legumain-responsive AuNPs (D&H-A-A&C) could passively target the glioma site and form in situ aggregates in response to legumain, leading to enhanced accumulation of doxorubicin (DOX) and hydroxychloroquine (HCQ) at the glioma site. HCQ could inhibit the DOX-induced cytoprotective autophagy and thus resensitize glioma cells to DOX. Parallelly, inhibiting autophagy could also inhibit the formation of autophagy-related vasculogenic mimicry (VM) by glioma stem cells. In vivo studies demonstrated that D&H-A-A&C possessed promising antiglioma effect. Moreover, cotreatment with anti-PD-L1 antibody was able to neutralize immunosuppressed glioma microenvironment and thus unleash antiglioma immune response. In vivo studies showed D&H-A-A&C plus anti-PD-L1 antibody could further enhance antiglioma effect and efficiently prevent recurrence. The effectiveness of this strategy presents a potential avenue to develop a more effective and more personalized combination therapeutic regimen for glioma patients.

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.

Expressed in macrophages and endothelial cells, the receptor for angiopoietin, tyrosine kinase with immunoglobulin and epidermal growth factor homology-2 (Tie2), is required for the reconstruction of blood vessels in tumor recurrence after chemotherapy. Thus, small therapeutic peptides that target and block Tie2 activity are promising as a therapeutic for the prevention of tumor relapse after chemotherapy. However, such small peptides often have low bioavailability, undergo rapid enzymatic degradation, and exhibit a short circulation half-life, making them ineffective in cancer therapy. Herein, we designed a dual-responsive amphiphilic peptide (mPEG1000-K(DEAP)-AAN-NLLMAAS) to modify the small peptide T4 (NLLMAAS) as a Tie2 inhibitor, endowing it with the ability to endure in circulation and specifically target tumor tissue. The ultimate nanoformulation (P-T4) releases T4 in response to the combination of the acidic tumor microenvironment and the presence of legumain, which is commonly overexpressed in tu...

Many antibody-drug conjugates (ADCs) have failed to achieve a sufficient therapeutic window in clinical studies either due to target-mediated or off-target toxicities. To achieve an additional safety level, a new class of antibody-prodrug conjugates (APDCs) directed against different targets in solid tumors is here described. The tumor-associated lysosomal endopeptidase legumain with a unique cleavage sequence was utilized for APDC metabolism. Legumain-activatable APDCs were as potent as their cathepsin B-activatable analogues. The peptide sequence susceptible to legumain cleavage was optimized for further discrimination of the formation of active metabolites within tumor cells versus healthy tissues, leveraging different tissue-specific legumain activities. Optimized APDCs with slow legumain-mediated conversion reduced preclinically the levels of active metabolite in healthy organs while retaining high activity against different TWEAKR- and B7H3-expressing tumors.

Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. It is ubiquitously expressed in various tissues and besides its involvement in immune response and other physiological processes, it was also shown to play a role in pathological states such as inflammation and cancer. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins derived from MDA-MB-231 cells using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed narrow cleavage specificity for P1 position combined with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events.

Epithelial-mesenchymal transition (EMT) is closely associated with the development of drug resistance. Lipid metabolism plays an important role in EMT. This work was to study the cholesterol-lowering drug simvastatin for reversing EMT-associated resistance to chemotherapy via lipid metabolism. Methods: The combination of simvastatin and paclitaxel was used to overcome the EMT-associated drug resistance. For dual-action on both cancer cells and tumor-associated macrophages (TAM), the tumor microenvironment-activatable multifunctional liposomes were developed for drug codelivery. The liposomes were modified with a hairpin-structured, activatable cell-penetrating peptide that is specifically responsive to the tumor-associated protease legumain. Results: It was revealed simvastatin can disrupt lipid rafts (cholesterol-rich domains) and suppress integrin-β3 and focal adhesion formation, thus inhibiting FAK signaling pathway and re-sensitizing the drug-resistant cancer cells to paclitaxel. Furthermore, simvastatin was able to re-polarize tumor-associated macrophages (TAM), promoting M2-to-M1 phenotype switch via cholesterol-associated LXR/ABCA1 regulation. The repolarization increased TNF-α, but attenuated TGF-β, which, in turn, remodeled the tumor microenvironment and suppressed EMT. The liposomal formulation achieved enhanced treatment efficacy. Conclusion: This study provides a promising simvastatin-based nanomedicine strategy targeting cholesterol metabolism to reverse EMT and repolarize TAM to treat drug-resistant cancer. The elucidation of the molecular pathways (cholesterol/lipid raft/integrin β3/FAK and cholesterol-associated LXR/ABCA1 regulation) for anti-EMT and the new application of simvastatin should be of clinical significance.

Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections1. Reactive oxygen species (ROS) can drive tumor formation2–4, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation, which is enhanced by the ROS-promoting bacterial oncoprotein CagA, dysregulates intracellular legumain processing and localization and decreases legumain activity in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between the precise oxidation of a host protein and tumorigenic signaling during bacterial infection and demonstrate the importance of oxidative post-translational modifications in tumor growth.

Butelase-1, an asparaginyl endopeptidase or legumain, is the prototypical and fastest known Asn/Asp-specific peptide ligase. It is highly useful for engineering and macrocyclization of peptides and proteins. However, certain biochemical properties and applications of naturally occurring and recombinant butelase-1 remain unexplored. Here we report methods to increase the yield of natural and bacterial expressed recombinant butelase-1 and how they can be used to improve the stability and activity of two important industrial enzymes, lipase and phytase, by end-to-end circularization. First, the yield of natural butelase-1 was increased 3-fold to 15 mg kg(-1) by determining its highest distribution which is found in young tissues, such as shoots. The yield of recombinantly-produced soluble butelase-1 was improved by promoting cytoplasmic disulfide folding, codon changes, and truncation of the N-terminal pro-domain. Natural and recombinant butelase-1 displayed similar ligase activity, physical stability, and salt tolerance. Furthermore, the processing and glycosylation sites of natural and recombinant butelase-1 were determined by proteomic analysis. Storage conditions for both forms of butelase-1, frozen or lyophilized, were also optimized. Cyclization of lipase and phytase mediated by either soluble or immobilized butelase-1 was highly efficient and simple, and resulted in increased thermal stability and enhanced enzymatic activity. Overall, improved production of butelase-1 can be exploited to improve the biocatalytic efficacy of lipase and phytase by end-to-end cyclization. In turn, ligase-improved enzymes could be a general and environmentally friendly strategy for producing more stable and efficient industrial enzymes. Ministry of Education (MOE) Nanyang Technological University Published version This work was supported by the Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) and a Nanyang Technological University internal funding-Synzymes and Natural Products Center (SYNC).

Over the past two decades, antibody drug conjugates (ADCs) and small molecule drug conjugates (SMDCs) have widely employed valine-citruline and related cathepsin-cleavable linkers due to their stability in plasma and their rapid cleavage by lysosomal cathepsins. However, a number of recent studies have illustrated that these linkers are subject to cleavage by exogenous enzymes such as Ces1C and neutrophil elastase, thus resulting in off-target release of drug. As such, there is a need to diversify the portfolio of ADC linkers in order to overcome nonspecific drug release. Rather than targeting cathepsins, we began with an "enzyme agnostic" screen in which a panel of 75 peptide FRET pairs were screened for cleavage in lysosomal extracts and in plasma. Unexpectedly, a series of Asn-containing peptides emerged from this screen as being cleaved far more quickly than traditional ValCit-type linkers while retaining excellent stability in plasma. Catabolism studies demonstrated that these linkers were cleaved by legumain, an asparaginyl endopeptidase that is overexpressed in a variety of cancers and is known to be present in the lysosome. MMAE-containing ADCs that incorporated these new linkers were shown to exhibit highly potent and selective cytotoxicity, comparable to analogous ValCit ADCs. Importantly, the Asn-containing linkers were shown to be completely stable to human neutrophil elastase, an enzyme thought to be responsible for the neutropenia and thrombocytopenia associated with ValCitPABC-MMAE ADCs. The legumain-cleavable ADCs were shown to have excellent stability in both mouse and human serum, retaining >85% of the drug after 1 week of incubation. Moreover, the corresponding small molecule FRET pairs exhibited

Tumour-associated macrophages (TAMs) represent an attractive cell target for anticancer therapy. However, selective and efficient targeting of TAMs remains difficult. Here, we constructed a novel dually functionalised nanoparticle platform (s-Tpep-NPs) by surface co-modification of nanoparticles (NPs) with tuftsin (Tpep) and legumain protease-sheddable polyethylene glycol 5k (PEG5k) to achieve selective targeted delivery to TAMs. The fluorescence resonance energy transfer experiment and in vitro cellular uptake assay confirmed that s-Tpep-NPs can responsively shed PEG5k and transform into active Tpep-NPs upon the cleavage of legumain that is overexpressed on TAM surfaces, which then promotes TAM phagocytosis through Fc receptor-mediated pathways. Owing to the shielding effect by legumain-sheddable PEG5k, s-Tpep-NPs can effectively decrease the Tpep-induced non-specific accumulation in mononuclear phagocyte system (MPS) organs during systemic circulation. Moreover, s-Tpep-NPs can significantly enhance the tumoural accumulation and improve the specificity and efficiency of targeting to TAMs, as compared with both controls of Tpep-NPs and non-sheddable ns-Tpep-NPs. Overall, this study provides a robust nanoplatform with a novel avenue for improved selectivity of targeted delivery to TAMs.

M2 tumor-associated macrophages (TAMs) play a pivotal role in cancer progression and therapy resistance. Inhibition of TAMs is of great significance to reshape the protumor environment to benefit therapeutic outcomes. In this work, we developed a novel TAMs and tumor cells dual-targeting nanoparticle (ATpep-NPs) system for cancer chemotherapy by integrating a docetaxel (DTX)-loaded nanocarrier and a multi-function peptide ATpep, which is composed of a phagocytosis-stimulating peptide-tuftsin (Tpep) fused with a substrate peptide-alanine-alanine-asparagine (AAN) of endoprotease legumain. In vitro protelytic and cellular uptake assays confirmed ATpep-NPs can be responsively activated into Tpep-NPs by cleavage of legumain that is overexpressed in both tumor cells and TAMs, which then promoted tumor cells internalization and TAMs phagocytosis through neuropilin-1/Fc receptor pathways. Due to AAN deactivation effect, ATpep-NPs can effectively decrease the Tpep-induced non-specific uptake by M1-polarized and normal macrophage during systemic circulation. Our results of in vivo experiments demonstrated ATpep-NPs outperformed Tpep-NPs in tumor and TAMs dual-targeting delivery efficiency with markedly enhanced efficacy against both tumor growth inhibition and TAMs depletion. Overall, this study offers a novel approach for development of multitargeted delivery vehicle for improved cancer chemotherapy.

Chronic pancreatitis (CP) is a major risk factor for pancreatic cancer; however, little is known about the pathogenic mechanisms underlying the development of CP. Legumain (Lgmn) has been linked to some chronic inflammatory diseases. The present study investigated the role of legumain in pancreatic fibrogenesis. We induced CP in wild type C57BL6 (WT), Lgmn-deficient (Lgmn−/−), Lgmnflox/flox and Lgmnflox/flox × LysMCre mice by intraperitoneal injection of caerulein for 4 weeks. Pancreata were collected and analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and histology. Pancreatic stellate cells and macrophages were isolated and studied using immunofluorescence, gelatin zymography, and enzyme-linked immunosorbent assay. The effects of inhibition of legumain were investigated in vivo by administration of the specific legumain inhibitor, RR-11a. Legumain was found to be upregulated in the serum and pancreatic tissues of mice with caerulein-induced CP. Mice with global and macrophage-specific legumain deficiency exhibited significantly reduced development of pancreatic fibrosis compared with control mice, based on pancreas size, histology, and expression of fibrosis-associated genes. Our results indicate that legumain promotes activation of pancreatic stellate cells and increases synthesis of extracellular matrix proteins via activation of matrix metalloproteinase-2(MMP-2), which hydrolyzes the transforming growth factor-β1 (TGF-β1) precursor to form active TGF-β1. Administration of RR-11a markedly attenuated pancreatic fibrosis in mice with CP. Deficiency or inhibition of legumain significantly reduces the severity of pancreatic fibrosis by suppressing activation of the TGF-β1 precursor. Our results highlight the potential of legumain as a novel therapeutic target for CP. • Legumain expression was markedly upregulated in CP mice. • Deletion of legumain attenuated pancreatic fibrosis in CP mice. • Legumain promotes fibrosis via MMP-2 activation, which hydrolyzed the TGF-β1 precursor to the active form. • Legumain is a potential therapeutic target for the management of CP.

The tumor microenvironment (TME) acts as an ecosystem that includes not only tumor cells, but also stromal cells such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). In addition, the abnormal extracellular environment (ECM), of which the mechanical forces are regulated by fibronectin (Fn) and collagen I, orchestrates tumorigenesis and progression by directly promoting invasion and cellular transformation of the ecosystem. Herein, we develop a novel peptide-modified liposome incorporated into doxorubicin (FnBPA5-AAN-Dox) as an ecological therapy system, which targets not only the cellular compartment but also non-cellular components of breast cancer. FnBPA5 is a Fn-binding peptide showing high affinity with relaxed Fn and collagen I in the ECM as well as α-SMA-expressing CAFs. However, the fast clearance by Fn-excreting organs such as the liver and spleen limits the accumulation of FnBPA5-Dox in the TME. The AAN peptide, which targets legumain overexpressed in the TAMs, could extend the circulation time and improve the therapeutic response as well as modulate the tumor immune microenvironment (TMIE). Given twice at an equivalent dose of 5 mg kg−1 intravenously, the multi-in-one ‘ecological therapy’ applied AAN-FnBPA5-Dox showed excellent antitumor efficacy in 4T1 breast cancer mice, and the tumor growth inhibition (TGI) is up to 98.20% compared with saline. Immunofluorescence, flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) results revealed that the dramatic improvement in antitumor efficacy can be attributed to the multifunctional targets of the drug delivery system.

Legumain enzyme is a well-conserved lysosomal cysteine protease and is over-expressed in many tumor cells and tumor stromal cells and exhibits higher protease activity under acidic conditions, such as in lysosomes and endosomes. Legumain enzyme-triggered drug delivery systems have demonstrated potential therapeutic values in cancer targeted therapy. To realize a more efficient delivery of anticancer therapeutic agents, we herein report a legumain/pH dual-responsive drug delivery system for enhancing site-specific controlled release of antitumor drugs. The carrier (named "DS-NA") is a hybrid vector constituting PEG-b-PBLA polymers, pH-responsive OAPI polymers, and legumain-sensitive peptide-doxorubicin prodrug decorated fluorescent carbon dots (CDs-C9-AANL-DOX). In tumor cells, DS-NA could disassemble rapidly in acidic environments, and then release doxorubicin through legumain digestion. Except as a drug vector, the drug release process from DS-NA could also be dynamically monitored by CLSM as the DOX was released from the surface of CDs through the AANL peptide linker digested by legumain, then transferred into the cell nucleus and exerted cytotoxicity, while the CDs themselves remained in the cytoplasm. As a control, the CDs-C9-DOX, which did not contain the AANL peptide linker, also still resided in the cytoplasm. Furthermore, in vivo studies show that DS-NA had a stronger inhibitory effect on tumor tissue with attenuated side effects to normal tissues than control nanoparticles or free drugs, which may be due to comprehensive effects including pH/legumain dual-triggered drug release, long blood circulation periods, and EPR effects. Together, a combination strategy of acid sensitivity and legumain enzyme sensitivity used for site-specific controlled release of drugs provides a novel method for enhanced and precise antitumor chemotherapy.

Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris. The pPICZα-LGMN expression plasmid was constructed and transformed into Pichia pastoris strain and positive recombinants were identified. Fermentation conditions were optimized and it was found that Legumain was most highly expressed under pH 6 culture conditions. In addition, the enzyme activity of the purified Legumain was tested using a fluorogenic substrate (Z-Ala-Ala-Asn-AMC) assay and the optimum pH for the autocatalytic activation of recombinant Legumain was very acidic at a pH value of 3. The recombinant protein was then used to screen a library of compounds and small molecule 1773 (Terramycin) was shown to effectively inhibit Legumain enzyme activity. These results indicate that the Pichia pastoris expression system can produce highly active recombinant Legumain protein allowing it to be used for High-throughput screening (HTS) applications.

Legumain an asparginyl endopeptidase expressed by both tumor cells and cells present in tumor microenvironment is an ideal therapeutic target for development of cancer therapies due to its correlation with high metastasis and invasion in various cancers. Microbial derivatives have demonstrated many pharmacological properties such as antioxidant, anti-inflammatory, anti tumor and immunostimulatory activities.In the current study, 541 microbial derivatives were screened for their potential to inhibit legumain using Lib dock .Out of 541 compounds screened we have identified 55 microbial derivatives which showed binding to legumain by docking. Molecular interaction analysis of top five docked derivatives revealed the interaction of derivatives with the catalytic residues of legumain. These compounds need to be further evaluated in vitro and in vivo for Legumain inhibition and ultimately cancer regression.

The lysosomal endoprotease legumain (asparaginyl endoprotease) has been proposed as a putative biomarker in prostate tumours, in which the enzyme is markedly overexpressed. Overexpression, coupled with highly selective specificity for cleavage of substrates at the C-terminus of asparagine (Asn) residues, make legumain an attractive biochemical target for potential diagnosis, prognosis and treatment. We report the design, synthesis, characterisation and preliminary evaluation of a new rhodamine-B (Rho-B)-labelled legumain peptide substrate probe5[Rho-Pro-Ala-Asn-PEG-AQ(4-OH)] and its selective targeting to lysosomes in PC3 prostate cancer cells. Probe5was efficiently activated by recombinant human legumain to afford the high quantum yield reporter fluorophore tripeptide4b(Rho-Pro-Ala-Asn-OH) with concomitant release of intense fluorescence. Furthermore, probe5was activated upon incubation with homogenates derived from fresh-frozen tissue material of prostatectomy specimens. Probe5represents a new viable biochemical tool for probing the activity of legumain with the potential to be used in ex vivo diagnostics in the cancer pathology laboratory.

Despite the great achievements of nanomedicines made in cancer chemotherapy, precise tumor targeting and deep penetration are still major challenges. Many nanomedicines can only passively accumulate near leaky site of tumor vessels through the enhanced permeability and retention (EPR) effect and cannot reach the deep region of tumor. To improve the tumor targeting, penetration and retention efficiency, an iRGD-mediated and enzyme-induced precise targeting gold nanoparticles system (iRGD/AuNPs-A&C) was developed by simply coadministering a tumor-homing penetration peptide iRGD (CRGDKGPDC) with a legumain responsive aggregable gold nanoparticle (AuNPs-A&C). In vitro, iRGD/AuNPs-A&C showed a consistent increase rate in size with AuNPs-A&C, suggesting that iRGD/AuNPs-A&C could also aggregate in the presence of legumain, which was in favor of the enhanced retention in tumor microenvironment. Meanwhile, iRGD/AuNPs-A&C showed higher 4T1 cells cellular uptake in vitro and presented higher penetration and accumulation in breast tumor in vivo than AuNPs-A&C, leading to enhanced tumor imaging efficacy and the improved chemotherapeutic effect to 4T1-bearing mice. These results suggested that the iRGD-mediated and enzyme-induced dual-functional nanoplatform was promising for the 4T1 tumor imaging and treatment.

Specific drug delivery to metastatic tumors remains a great challenge for antimetastasis therapy. We herein report a bioengineered macrophage-based delivery system (LD-MDS) that can be preferentially delivered to lung metastases and intelligently transformed into nanovesicles and secondary nanovesicles for antimetastasis therapy. LD-MDS was prepared by anchoring a legumain-specific propeptide of melittin (legM) and cytotoxic soravtansine (DM4) prodrug onto the membrane of living macrophages. LD-MDS is responsively activated by legumain protease and converted into DM4-loaded exosome-like nanovesicles (DENs), facilitating efficient internalization by metastatic 4T1 cancer cells and considerable cell death. Afterward, the damaged 4T1 cells can release secondary nanovesicles and free drug molecules to destroy neighboring cancer cells. In vivo, LD-MDS displays superior targeting efficiency for lung metastatic lesions with diameters less than 100 μm and remarkably inhibits lung metastasis. This study provides a new opportunity to explore endogenous macrophages as living drug delivery vehicles with controlled drug release to target metastatic lung tumors.

Legumain is one of the cysteine proteases which can serve as an essential indicator for cancer diagnosis. Near-infrared (NIR) nanoprobes with fluorescence "Turn On" property are advantageous in cancer diagnosis. However, to the best of our knowledge, using a completely organic NIR nanoprobe to image legumain activity either in vitro or in vivo has not been reported. Herein, employing a CBT-Cys click condensation reaction, we used a rationally designed NIR probe Cys(StBu)-Ala-Ala-Asn-Lys(Cy5.5)-CBT (1) to synthesize its nanoprobes 1-NPs with self-quenched fluorescence. Cell and animal experiments indicated that our nanoprobes were able to specifically image legumain activity in living cells and tumors with a NIR fluorescence "Turn On" manner. We envision that the nanoprobes could be applied for the diagnosis of legumain-related diseases in the near future.

Multidrug resistance proteins (MDRPs), which are implicated in the mediation of multidrug resistance in tumors, represent the main obstacle to successful chemotherapy. As curcumin (Cur) exerts inhibitory effects on both the expression and function of MDRPs, a nanocarrier for the co-delivery of Cur and doxorubicin (DOX) was prepared to overcome MDR tumors through their synergistic effects. Owing to the overexpression of legumain in tumors, the release profile of DOX from this nanocarrier was designed to be legumain modulated, which was achieved by bridging DOX to a basic material (chitosan) with a legumain-sensitive peptide. Compared with nanoparticles that only contain DOX, the coadministration of DOX and Cur significantly inhibited multidrug resistance (P < 0.05) in a multidrug-resistant cancer cell model (MCF-7/ADR cell line), with cytotoxicity to normal cells (L929 cell line). Such inhibition could be ascribed to the increased DOX accumulation in the MCF-7/ADR nucleus. The co-delivery system exhibited good anticancer effects through prolonged circulation time, improved tumor-targeting efficiency, elevation of the tumor inhibition activity, and the suppression of MDRP expression. These data revealed the enormous potential of this co-delivery system for cancer therapy, especially in the later stages where multidrug resistance may develop.

Two distinct macrophage phenotypes contribute to kidney injury and repair during the progression of renal interstitial fibrosis; proinflammatory (M1) and antiinflammatory (M2) macrophages. Legumain, an asparaginyl endopeptidase of the cysteine protease family, is overexpressed in macrophages in some pathological conditions. However, the macrophage subtype and function of macrophage-derived legumain remains unclear. To resolve this we tested whether M2 macrophages contribute to the accumulation of legumain in the unilateral ureteral obstruction model. Legumain-null mice exhibited more severe fibrotic lesions after obstruction compared with wild-type control. In vitro, IL4-stimulated M2 polarization led to the overexpression and secretion of legumain. The levels of fibronectin and collagen I/III, major components of the extracellular matrix, were reduced in the conditioned medium of TGF-β1-stimulated tubular epithelial cells or fibroblasts after treatment with legumain or conditioned medium from IL4-stimulated macrophages. Administration of the legumain inhibitor RR-11a exacerbated fibrotic lesions following obstruction. Therapeutically, adoptive transfer of legumain-overexpressing macrophages or IL4-stimulated macrophages ameliorated the deposition of collagen and fibronectin induced by ureteral obstruction, either in the wild-type mice or in lgmn–/– mice. Thus, M2 macrophages overexpress and secret legumain and legumain mediates the anti-fibrotic effect of M2 macrophages in obstructive nephropathy.

Background Asparaginyl endopeptidase, also known as legumain (EC 3.4.22.34) shows strong activity in the mouse kidney. Legumain is also highly expressed in tumors. DJ-1/PARK7 is a Parkinson's disease- and cancer-associated protein. DJ-1 is a coactivator of various transcription factors. Recently, we reported that transcription of the legumain gene is regulated by p53 through DJ-1. Methods We measured the secretion levels of legumain in a conditioned medium of DJ-1 knockout cells and in serum from DJ-1 knockout mice using Western blotting and ELISA. We performed immunocytochemical staining of legumain to examine the localization of legumain in DJ-1-knockout cells. Results We found that the secretion levels of legumain were increased in the conditioned medium of DJ-1-knockout cells and in serum from DJ-1-knockout mice. Dot structures of legumain were also increased in DJ-1-knockout cells. Conclusion The results suggest that legumain secretion from DJ-1-knockout cells and in mice increases through its increased expression and accumulation in membrane-associated vesicles.

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering. Ministry of Education (MOE) Nanyang Technological University Published version This research was supported by an Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to J. P. T. and internal funding from Nanyang Technological University, Synzymes and Natural Products Center (SYNC).

Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aβ production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD.

Emotional and cognitive impairment has been recognized as a central feature of depression, which is closely related to hyperfunction of the hypothalamic-pituitary-adrenal (HPA) axis caused by down-regulation of glucocorticoid receptor (GR) expression in patients. A decrease in GR expression can cause pathological changes and lead to the impairment of synaptic plasticity. Legumain, a lysosomal cysteine protease, plays an important role in neurological diseases. It is reported that legumain activates the MAPK signaling pathway, which modifies the GR. Therefore, we hypothesize that regulation of the GR by legumain plays a crucial role in the pathological process of depression. The relationships between legumain, GR, synaptic plasticity and emotional and cognitive deficits were explored in this study. The results demonstrated that repeated corticosterone (CORT) injections (3 weeks) induced emotional and cognitive deficits in mice, based on behavioral experiments and the detection of synaptic plasticity. Furthermore, CORT injections decreased the expression of hippocampal synapse-related proteins, cell density and dendritic spine density in the hippocampus, accompanied by increased protein expression in the MAPK signaling pathway and decreased expression of the GR. In conclusion, our results demonstrated that legumain knockout up-regulated expression of the GR by reducing protein expression in the MAPK signaling pathway, thereby improving hippocampal synaptic plasticity as well as the emotional and cognitive impairment of model mice. This suggests that legumain may be an effective therapeutic target for emotional and cognitive deficits.

Neuroblastoma (NB) is a highly malignant solid tumor in children. The cysteine endopeptidase legumain is expressed in adult solid tumors, but its expression in NB has not been examined. In this study, we assayed legumain expression in two NB cell lines and in microarrays of tumor tissues collected from 46 children with undifferentiated NB, differentiated NB, and ganglioneuroblastoma. Correlation analyses showed that legumain was expressed in all NB cell lines tested and that expression correlated with the degree of tumor differentiation. The efficacy, specificity, and toxicity of EMC-AANL-DOX, a novel doxorubicin-based legumain-activated prodrug, were then evaluated in mouse model of NB. Compared with DOX, EMC-AANL-DOX showed greater inhibition of tumor growth and a lower toxicity at high doses. Neither leukocyte or platelet counts nor renal function or cardiac anatomy differed significantly between the EMC-AANL-DOX and control groups (p > .05), suggesting that the prodrug caused minimal bone marrow depression and did not induce renal or cardiac damage. The good specificity and efficacy of EMC-AANL-DOX and low toxicity recommend its use in the treatment of NB. Correlation analyses of NB differentiation and legumain expression may reveal a novel anti-tumor-related functions of the drug and a new strategy for the treatment of pediatric solid tumors.

SUMMARYThe present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35–39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

Asparagine endopeptidase (AEP), also called legumain, is highly expressed in various solid tumors, promoting cancer cell invasion, migration, and metastasis. It has been proposed to be a prognostic marker and therapeutic target for cancer treatment. However, an effective nonpeptide, small-molecule inhibitor against this protease has not yet been identified. Here we show that a family of xanthine derivatives selectively inhibit AEP and suppress matrix metalloproteinase (MMP) cleavage, leading to the inhibition of cancer metastasis. Through structure-activity relationship (SAR) analysis, we obtained an optimized lead compound (38u) that represses breast cancer invasion and migration. Chronic treatment of nude mice, which had been inoculated with MDA-MB-231 cells, with inhibitor 38u via oral administration robustly inhibits breast cancer lung metastasis in a dose-dependent manner, associated with blockade of MMP-2 by AEP. Therefore, our study supports that 38u might act as a potent and specific AEP inhibitor useful for cancer treatment.

Platinum-based drugs are widely used as a master key in the multiple cancer therapies, while limited by the adverse side effects during the in vivo circulation. To settle the aforementioned problems, we prepare a series of polymer-platinum conjugates by utilizing the “stealth” polyethylene glycol and legumain-cleavable oligopeptide. We select MGC803 cells overexpressing the legumain and MKN28 cells negatively expressing the legumain as well as the normal stomach cells to compose the test groups. In vitro cytotoxicity studies indicate the polymer-platinum conjugates with the assistance of Ala-Ala-Asn tripeptide can effectively inhibit the growth of MGC-803 cells than control groups. Meanwhile, the MPEG polymer show better anticancer ability than 4-arm-PEG polymer. However, in the group of MKN28 cells, both polymer-platinum conjugates express weak cytotoxicity due to lacking of the mediation of legumain. Hence, the synthesized polymer-platinum conjugates exhibiting great potential in avoiding the side effects and enhancing the therapy ability will be valuable for the platinum-cancer therapy.

Proteolysis is an important process for development and germination of cereal seeds. Among the many types of proteases identified in plants are the cysteine proteases (CPs) of the papain and legumain families, which play a crucial role in hydrolysing storage proteins during seed germination as well as in processing the precursors of these proteins and the inactive forms of other proteases. Moreover, all of the tissues of cereal seeds undergo progressive degradation via programed cell death, which is integral to their growth. In view of the important roles played by proteases, their uncontrolled activity could be harmful to the development of seeds and young seedlings. Thus, the activities of these enzymes are regulated by intracellular inhibitors called phytocystatins (PhyCys). The phytocystatins inhibit the activity of proteases of the papain family, and the presence of an additional motif in their C-termini allows them to also regulate the activity of members of the legumain family. A balance between the levels of cysteine proteases and phytocystatins is necessary for proper cereal seed development, and this is maintained through the antagonistic activities of gibberellins (GAs) and abscisic acid (ABA), which regulate the expression of the corresponding genes. Transcriptional regulation of cysteine proteases and phytocystatins is determined by cis-acting elements located in the promoters of these genes and by the expression of their corresponding transcription factors (TFs) and the interactions between different TFs.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions. Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component ( Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.