Activity Of Cathepsin Research Articles

Autophagy-lysosomal pathway impairment and cathepsin dysregulation in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by neuronal loss, attributed to amyloid-beta (Aβ) aggregation and accumulation. The autophagy-lysosomal pathway, including cathepsins B and D, is crucial for protein degradation and clearance, but it is impaired in some diseases. This review summarizes current knowledge on the dysregulation of this pathway in AD. Accumulating evidence suggests that Aβ overload impairs autophagy-lysosomal function and cathepsin activity, exacerbating Aβ accumulation and neurodegeneration. However, the precise mechanisms underlying these interactions remain elusive. Despite these challenges, targeting the lysosomal pathway emerges as a promising therapeutic strategy, and a comprehensive understanding of the autophagy-lysosomal system is essential to develop effective interventions for AD.

The Activation of Endogenous Proteases in Shrimp Muscle Under Water-Free Live Transport.

Water-free transportation (WFT) causes shrimp (Penaeus vannamei) flesh quality deterioration. However, the roles of endogenous protease-induced protein hydrolysis have been neglected in the research. In the present study, calpain zymography, gelatinase zymography, the hematoxylin-eosin staining method, and other methods were applied to investigate the response of various endogenous proteases (cathepsin, calpain, and gelatinase), the myofibril fragmentation index (MFI), and the microscopic morphology of shrimp muscle during WFT in comparison with the shrimp under the conventional water transportation strategy (WT). The results showed that the total activity of proteases in shrimp muscle increased significantly (p ≤ 0.05) after simulated transportation. Cathepsins and gelatinases were activated during WFT. No significant (p > 0.05) changes of the activity of caspase-3 and the muscle cell apoptosis rate were detected in shrimp muscle cells after WFT. In addition, the MFI increased and the gap among muscle fiber bundles enlarged after WFT. Compared with WFT, no significant (p > 0.05) effect on the activities of calpain, gelatinase, and caspase-3 in the muscle of shrimp was found after WT, and only the activity of cathepsin L significantly increased (p ≤ 0.05). Based on the findings, we concluded that the activation of various endogenous proteases was induced during WFT.

Profiling Cysteine Proteases Activities in Neuroinflammatory Cells.

A new activity-based probe (ABP) of cysteine proteases (FGA139) has been designed and synthesized. The design of the ABP has been done based upon the chemical structure of an irreversible inhibitor of cysteine proteases by attaching a bodipy fluorophore at the N-terminus of the peptide backbone. The synthetic route of the probe has a metathesis and a "click" reaction as key steps. Although some studies have been reported about the role played by cysteine proteases in neurodegenerative diseases, there are not definitive conclusions. The obtained ABP has been employed as a chemical tool to profile activities of cysteine proteases cathepsins B, L, and calpain in neurodegenerative cell models through confocal imaging. Colocalization of the probe to specific antibodies of the proteases and competitive experiments with non-fluorescent inhibitors confirm the specificity of the ABP. From a theranostic perspective, our findings strongly suggest that FGA139 exhibits a protective role in various cell lines against oxidative stress or pro-inflammatory toxicity and it effectively attenuates macrophage activation triggered by LPS.

The modulatory effects of Lonicera caerulea L. extract and omega-3 on sarcopenia via regulating PI3K and FOXO signaling pathways in rats

Introduction: Sarcopenia is an inflammatory disease caused by a disruption of muscle homeostasis. Lonicera caerulea L. (Haskap berry) (HB) extract and omega-3 (ω−3) possess antioxidant and anti-inflammatory activities. This study assesses the potential ameliorating effects of HB extract and ω−3 supplementation on dexamethasone (DEXA)-induced sarcopenia. Methods: Rats were divided into five groups; the negative control group was injected with saline (i.p.); groups 2, 3, 4, and 5 were injected with DEXA (2 mg/kg/d, i.p.); groups 3 and 4 also received 400 mg/kg/d and 100 mg/kg/d of HB extract and ω−3, respectively, while group 5 received both treatments daily for 21 days. The ameliorative effects of treatments were investigated by measuring lysosomal proteolytic enzyme cathepsin activity, phosphatidylinositol 3 kinase (PI3K) activity, nuclear factor kappa beta (NF-κB) level, and heme oxygenase-1 (HO-1) activity. The gene expression levels of muscle ring-finger protein (MuRF) and forkhead box O (FOXO) transcription factor were also evaluated. Biochemical and histological examinations were done on muscle tissues. Results: DEXA caused a significant elevation (P<0.05) in NF-κβ level and cathepsin activity in muscle tissue. Also, it significantly upregulated the muscle atrophy markers. Meanwhile, PI3K and HO-1 activities were significantly reduced. HB extract and ω−3 administration significantly (P≤0.05) reversed these effects and downregulated the mRNA expression levels of MuRF and FOXO. Also, the histopathological examination confirmed these results. Conclusion: The current study proved the ameliorative effects of HB extract and ω−3 on sarcopenia parameters. Therefore, they might be potential therapeutic agents for myopathy/sarcopenia.

Immunohistochemical Evaluation of Cathepsin B, L, and S Expression in Breast Cancer Patients.

Cysteine cathepsins are proteases that play a role in normal cellular physiology and neoplastic transformation. Elevated expression and enzymatic activity of cathepsins in breast cancer (BCa) indicates their potential as a target for tumor imaging. In particular cathepsin B (CTSB), L (CTSL), and S (CTSS) are used as targets for near-infrared (NIR) fluorescence imaging (FI), a technique that allows real-time intraoperative tumor visualization and resection margin assessment. Therefore, this immunohistochemical study explores CTSB, CTSL, and CTSS expression levels in a large breast cancer patient cohort, to investigate in which BCa patients the use of cathepsin-targeted NIR FI may have added value. Protein expression was analyzed in tumor tissue microarrays (TMA) of BCa patients using immunohistochemistry and quantified as a total immunostaining score (TIS), ranging from 0-12. In total, the tissues of 557 BCa patients were included in the TMA. CTSB, CTSL, and CTSS were successfully scored in respectively 340, 373 and 252 tumors. All tumors showed CTSB, CTSL, and/or CTSS expression to some extent (TIS > 0). CTSB, CTSL, and CTSS expression was scored as high (TIS > 6) in respectively 28%, 80%, and 18% of tumors. In 89% of the tumors scored for all three cathepsins, the expression level of one or more of these proteases was scored as high (TIS > 6). Tumors showed significantly higher cathepsin expression levels with advancing Bloom-Richardson grade (p < 0.05). Cathepsin expression was highest in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2(HER2)-positive and triple-negative (TN) tumors. There was no significant difference in cathepsin expression between tumors that were treated with neoadjuvant systemic therapy and tumors that were not. The expression of at least one of the cysteine cathepsins B, L and S in all breast tumor tissues tested suggests that cathepsin-activatable imaging agents with broad reactivity for these three proteases will likely be effective in the vast majority of breast cancer patients, regardless of molecular subtype and treatment status. Patients with high grade ER-negative, HER2-positive, or TN tumors might show higher imaging signals.

A novel cystatin in Psoroptes ovis var. cuniculi: molecular characterization, serodiagnostic potential, and its anti-inflammatory property on rabbit peripheral blood mononuclear cells

BackgroundThe ectoparasite Psoroptes ovis var. cuniculi causes substantial economic losses to the global rabbit industry. Currently, microscopy for identifying Psoroptes mite in skin scrapings, as the “diagnosis gold standard,” remains a challenge owing to its poor sensitivity in detecting low-level and/or early stage mite infestations. Additionally, Psoroptes infestations rapidly trigger cutaneous inflammation, thus the mites might produce some molecules to deal with the harmful effects of inflammation for their long-time survival on the host skin, but these molecules are still mostly unknown.MethodsTo seek a sensitive diagnostic method and illuminate the new antiinflammatory molecules, we characterized a novel cystatin of P. ovis var. cuniculi (PsoCys) using bioinformatics and molecular biology methods.ResultsThe results showed that PsoCys comprised the classical features of the type II cystatin superfamily including an N-terminal glycine residue, a central QXVXG motif, and a C-terminal LW motif. In mixed stages of mites, the transcription level of PsoCys was significantly higher in “fed” mites than in “starved” mites (P < 0.001), and among the different life-cycle stages of “fed” mites, the expression of PsoCys was higher in adult males than in larva, nymph, and adult females (P < 0.001). The established indirect ELISA based on recombinant PsoCys (rPsoCys-iELISA) presented 95.4% sensitivity and 95.7% specificity. The area under the receiver operating characteristic curve (AUC) for this method was 0.991, indicating its excellent diagnostic performance. Moreover, rPsoCys-iELISA had advantages over microscopy for detecting low-level and/or early stage mite infestations (90% versus 40% in artificial infestation cases at 3 weeks post-infestation; 61.9% versus 22.6% in clinical cases). In addition, rPsoCys could inhibit the activity of papain and cathepsin B in vitro, and significantly suppressed mRNA levels of toll-like receptors (TLR 1, 2, 4, and 6) and downstream molecules (NF-κB, p38, MyD88, IL-10, and IFN-γ) in LPS-stimulated rabbit PBMCs, indicating its anti-inflammatory property.ConclusionsOur findings indicated that PsoCys was a novel type II cystatin of Psoroptes mites, and it served as a potential serological diagnostic antigen for detecting low-level and/or early stage mite infestations, as well as a novel anti-inflammatory molecule of Psoroptes mites.Graphical abstract

385. TRANSLATING MOLECULAR-TARGETING FLUORESCENT PROBES IN OESOPHAGEAL CANCER PRE-CLINICAL MODELS

Abstract Background Intra-operative molecular imaging (IMI) is an emerging field that utilises tumour-targeting fluorescent probes to improve oncological outcomes. A novel class of probe known as quenched activity-based probes (qABP), developed to measure enzyme activity in-vitro, has been repurposed to target tumour-expressed proteases known as cathepsins. Unlike other tumour-targeting probes, fluorescence is inhibited up until the qABP covalently bonds to cathepsins, releasing an inhibitory quencher which results in fluorescence. This may improve the contrast between normal tissue and tumour. Our laboratory group sought to obtain pre-clinical evidence to eventually translate qABP’s into the operating room for oesophageal, junctional and gastric cancer. Methods Cathepsin activity was screened in cell lines from the normal oesophagus, Barrett’s metaplasia, oesophageal adenocarcinoma (OAC), squamous cell carcinoma (SCC) and gastric adenocarcinoma (GAC) with two qABP’s known as BMV109 and VGT309. We additionally screened patient biopsies from oesophageal, junctional and gastric cancers. Cell line and tissue samples were analysed using in-gel fluorescence (Amersham Typhoon®) and Western blot. In-vivo, cell line xenografts were established in NSG mice using subcutaneous and orthotopic models. Tumour-bearing mice were then injected with VGT-309 and then imaged at specific timepoints using the IVIS® spectrum imager. Results Cathepsin activity was found in all oesophago-gastric cell lines and selectivity proven with a cathepsin inhibitor. Matched biopsies were collected from patients prior before and after neoadjuvant chemo-radiotherapy. Baseline tumour biopsies exhibited significantly higher cathepsin activity than normal biopsies, with these differences preserved in biopsies collected after chemo-radiotherapy (p &amp;lt;0.001). In-vivo, OAC and GAC xenografts were identified in NSG mice as early as 12 hours post injection of VGT-309, exhibiting a 2.5-fold increase in fluorescence compared to normal background stomach. Bio-distribution analysis demonstrated that VGT-309 accumulated in liver and kidney, but less so in the murine oesophagus and stomach. Conclusion Translating qABP’s into the operating room has the potential to detect early cancers in Barrett’s metaplasia, reduce positive margin rates and identify lymph node metastasis. Our research group continues to investigate qABP’s, including determining their sensitivity to detect lymph node metastasis with the aim of translating this technology into a phase 1 clinical trial.

Hydroxyl Radical-Induced Oxidation on the Properties of Cathepsin H and Its Influence in Myofibrillar Proteins Degradation of Coregonus peled In Vitro.

The most frequently occurring protein modification in fish postmortem is oxidization, which further affects meat quality through multiple biochemical pathways. To investigate how hydroxyl radicals affect the structure of cathepsin H and its ability to break down myofibrillar proteins in Coregonus peled, cathepsin H was oxidized with 0, 0.1, 0.5, 1, 5, and 10 mM H2O2 and subsequently incubated with isolated myofibrillar proteins. The results showed that as the H2O2 concentration increased, the carbonyl and sulfhydryl contents of cathepsin H significantly increased and decreased, respectively. There were noticeable changes in the α-helix structures and a gradual reduction in UV absorbance and fluorescence intensity, indicating that oxidation can induce the cross-linking and aggregation of cathepsin H. These structural changes further reduced the activity of cathepsin H, reaching its lowest at 10 mM H2O2, which was 53.63% of the activity at 0 mM H2O2. Moreover, desmin and troponin-T all degraded at faster rates when cathepsin H and myofibrillar proteins were oxidized concurrently as opposed to when cathepsin H was oxidized alone. These findings provide vital insights into the interaction mechanism between oxidation, cathepsin H, as well as myofibrillar protein degradation, laying a groundwork for understanding the molecular mechanisms underlying changes in fish meat quality after slaughter and during processing.

Functional analysis of novel cystatins from Haemaphysalis doenitzi and evaluation of their roles in cypermethrin and λ-cyhalothrin resistance

Currently, the primary strategy for tick control relies on chemical agents. Pyrethrins, which are botanically derived compounds, have demonstrated efficacy in controlling ticks without posing a risk to human or animal health. However, research into pyrethrins' metabolic mechanisms remains sparse. Cystatin, as a reversible binding inhibitor of cysteine protease, may be involved in the initiation of pyrethrin detoxification of Haemaphysalis doenitzi. In this study, two novel cystatins were cloned, HDcyst-3 and HDcyst-4, the relative expression of which was highest in the Malpighian tubules compared with the tick midguts, salivary glands, and ovaries. Prokaryotic expression and in vitro studies revealed that cystatins effectively inhibit the enzymatic activities of cathepsins B and S. RNAi results showed that the reduction of cystatins significantly decreased the engorgement weight, egg mass weight, and egg hatching rate of adult female ticks, and prolonged feeding time by two days. The control rate of rHDcyst-3 and rHDcyst-4 protein vaccination against female adults were 55.9% and 63.2%, respectively. In addition, the tick immersion test showed that cypermethrin and λ-cyhalothrin had significant acaricidal effects against adult unfed H. doenitzi. The qPCR result indicated that compared with the control group, the expression of HDcyst-3 and HDcyst-4 was markedly decreased in the sublethal cypermethrin and λ-cyhalothrin group at LC50. Enzyme activity showed that cypermethrin and λ-cyhalothrin could significantly induce the activities of glutathione S-transferase (GST), carboxylesterase (CarE), and acetylcholinesterase (AchE). The aforementioned results provided indirect evidence that cystatin plays an important role in pyrethrin detoxification and provides a theoretical basis for future acaricide experiments and pest management.

A dual inhibitor of PIP5K1C and PIKfyve prevents SARS-CoV-2 entry into cells

The SARS-CoV-2 pandemic has had an unprecedented impact on global public health and the economy. Although vaccines and antivirals have provided effective protection and treatment, the development of new small molecule-based antiviral candidates is imperative to improve clinical outcomes against SARS-CoV-2. In this study, we identified UNI418, a dual PIKfyve and PIP5K1C inhibitor, as a new chemical agent that inhibits SARS-CoV-2 entry into host cells. UNI418 inhibited the proteolytic activation of cathepsins, which is regulated by PIKfyve, resulting in the inhibition of cathepsin L-dependent proteolytic cleavage of the SARS-CoV-2 spike protein into its mature form, a critical step for viral endosomal escape. We also demonstrated that UNI418 prevented ACE2-mediated endocytosis of the virus via PIP5K1C inhibition. Our results identified PIKfyve and PIP5K1C as potential antiviral targets and UNI418 as a putative therapeutic compound against SARS-CoV-2.

Cystatin F Depletion in Mycobacterium tuberculosis-Infected Macrophages Improves Cathepsin C/Granzyme B-Driven Cytotoxic Effects on HIV-Infected Cells during Coinfection.

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.

Effects of lysosomal Fe2+ on mitochondria apoptosis and muscle tenderness in postmortem Tibetan sheep meat

SummaryIn this work, deferoxamine (DFO) was injected into Tibetan sheep meat (distilled water as control) to reveal the effects of lysosomal Fe2+ on mitochondrial apoptosis and muscle tenderness. The reactive oxygen species (ROS) content, lysosomal membrane stability, cathepsins activity, mitochondrial membrane permeability transition pore (MPTP), mitochondrial membrane potential (MMP), cytochrome c (Cyt‐c) reduction level, caspase activity, myofibril fragmentation index (MFI) and shear force of Oula Tibetan sheep meat were measured at different ageing times. The results indicated that the ROS content, cathepsins activity, MPTP opening degree, lysosomal membrane stability, caspase‐3/9 activity and MFI of Tibetan sheep meat were significantly decreased after DFO treatment, whilst MMP, shear force and Cyt‐c reduction levels were noticeably increased. Furthermore, correlation analysis and principal component analysis showed that mitochondrial damage was positively correlated with meat tenderness. In sum, the study suggests that lysosomal Fe2+ may promote mitochondrial apoptosis and improve the tenderness of Tibetan sheep meat.

Ultrasound combined with post-mortem aging enriches antioxidant peptides in Muscovy ducks

To investigate the impact of ultrasound in conjunction with post-mortem aging on antioxidant peptides in Muscovy ducks, this study was conducted under appropriate ultrasound conditions (20 min, 20 kHz, 1050 W), followed by post-mortem aging at 4 °C for 6, 12, 24,72 and 168 h. The analysis focused on the peptide content and antioxidant activity of Muscovy ducks. The results revealed that the peptide content reached the highest of 14.02 mg/g of meat following 72 h post-mortem aging combined with ultrasound treatment; an increase of 1.20 mg/g of meat of peptides was observed compared with the control. Moreover, the antioxidant activity also reached its peak (DPPH· scavenging efficacy was 47.38%). The sarcomeres almost completely disappeared after ultrasound treatment, the M and Z lines darkened, and the activities of cathepsins B, D, H, and L and calpain proteases increased. Gel filtration chromatography and reversed-phase high-performance liquid chromatography were utilized to isolate the peptides with the most potent antioxidant activity. Nine of the sequenced peptides were deemed reliable. ENHHCCC exhibited the highest antioxidant activity. Therefore, ultrasound in conjunction with post-mortem aging is a promising method to enrich antioxidant peptides in Muscovy duck.

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Tofacitinib Regulates Endostatin via Effects on CD147 and Cathepsin S.

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin' s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

Late-onset Krabbe disease presenting as spastic paraplegia - implications of GCase and CTSB/D.

Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). Additionally to cerebral MRI, protein structural analyses of the β-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of β-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.

Involvement of Endolysosomes and Aurora Kinase A in the Regulation of Amyloid β Protein Levels in Neurons.

Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer's disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aβ) generating enzyme β-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aβ degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aβ. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aβ. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aβ and extracellular amyloid plaque formation.

Rapamycin and Starvation Mitigate Indomethacin-Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

Synthesis of a Novel Hemicucurbituril and Its Inhibitory Activity of Cathepsin L

Synthesis of a Novel Hemicucurbituril and Its Inhibitory Activity of Cathepsin L

Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.