Lysosomal dysfunction and elevated lysosomal pH are hallmark features of age-related neurodegenerative diseases including Age-related Macular Degeneration (AMD), Alzheimer's Disease (AD), and Parkinson's Disease (PD). Restoring lysosomal acidity is important for maintaining enzymatic degradation, preventing protein aggregation, and reducing cellular waste accumulation in degenerating tissues. Acidic nanoparticles represent a promising therapeutic strategy to normalize lysosomal pH; however, accurate monitoring of their delivery, retention, and dosage is critical for rigorous evaluation. To address this, we developed fluorescently labeled poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles conjugated with Cyanine3 amine (Cy3). Nanoparticle uptake was systematically optimized, achieving over 90% delivery to lysosomes of induced pluripotent stem cell-derived retinal pigment epithelial (iPS-RPE) cells. Uptake rates varied among adjacent cells. Once internalized, nanoparticles demonstrated remarkable stability, with no detectable change in concentration, distribution, or size for at least 28 days. iPS-RPE cells exhibited higher nanoparticle internalization compared to the ARPE-19 cell line and optic nerve head astrocytes. The capacity of the nanoparticles to restore function to stressed lysosomes was confirmed by their ability to reacidify lysosomes, restore cathepsin B activity and increase levels of active cathepsin D. The nanoparticles also reduced levels of LC3II in astrocytes treated with chloroquine, indicating they can also restore autophagy rates. In summary, this study demonstrates the value of Cy3 labeling for enhanced nanoparticle tracking to lysosomes. The findings also identify PLGA nanoparticles as powerful tools for restoring degradative lysosomal function and autophagy in cells undergoing lysosomal stress.

TOF-probe-based mass cytometry reveals individual protease activity as an important driver of immune cell differentiation and function.

Analysis of "soft flesh"-affected Atlantic mackerel based on a systematic study of histology and protein structure.

In Brazil, the slaughter of female cattle, particularly older animals, has increased substantially. However, meat from this category presents limitations in terms of quality, mainly because of its toughness resulting from a high concentration of cross-links between collagen fibers. The aging process is an effective strategy to improve the quality of meat in this category because it stimulates the activity of cathepsins, which are enzymes responsible for collagen degradation. Calcium chloride (CaCl2) injection, in addition to activating calpains, can destabilize lysosomal membranes, releasing cathepsins and enhancing the effects of aging. Additionally, freezing inhibits calpastatins, which are natural inhibitors of calpains, resulting in increased activity of these proteolytic enzymes. Thus, the present study aimed to evaluate the effect of CaCl2 injection, in combination with freezing prior to wet aging, on the meat quality of cull cows. Meat samples injected with CaCl2 showed higher b* values. In the samples that were not previously frozen, higher L* values were recorded after 14 days of aging. At 3 days of aging, total collagen content was higher in previously frozen samples, whereas, at 14 days, no effect of freezing was observed. Under the conditions of this study, CaCl2 injection, whether combined with freezing or not, minimally affected the quality of aged cull cow meat. Furthermore, prior freezing can be avoided because it negatively impacts meat color. However, when freezing is already part of the industrial process, 14 days of aging has the potential to counteract its adverse effects. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Simultaneous detection of multiple cathepsin activities by peptide microarray-based metal-enhanced fluorescence assay

The Aurora B inhibitor ZM-447439 induces caspase-independent necrosis-like death in v-Src oncogene-expressing cells via accumulation of extra-lysosomal cathepsin B.

Avoiding lysosomal degradation is vital to the success of intracellular pathogens. The Gram-negative bacterium Coxiella burnetii and protozoan parasites of the Leishmania genus are unique in being able to replicate within the mature phagolysosomal compartment of host cells, though the exact mechanisms utilized to withstand this hostile environment are not clearly defined. We recently reported that C. burnetii removes the lysosomal protease cathepsin B during infection of mammalian cells. Here, we aimed to determine if this virulence strategy was also employed by the intralysosomal pathogen, Leishmania mexicana. In contrast to C. burnetii, decreases in the activity of specific cathepsins were not detected in L. mexicana-infected host cells as determined using immunoblotting and protease activity-based probes. Co-infection of THP-1 macrophage-like cells with both pathogens resulted in a proteolytic and secretory phenotype consistent with C. burnetii infection, suggesting that C. burnetii-induced remodeling of the lysosome is not influenced by L. mexicana. The host cell proteome and secretome of L. mexicana-infected cells were defined using mass spectrometry. This confirmed that, unlike C. burnetii, L. mexicana does not induce increased abundance of lysosomal proteins either intracellularly or in the extracellular milieu. Collectively, this study reveals that although C. burnetii and L. mexicana reside in a phagolysosomal intracellular niche, they employ divergent mechanisms to survive within this hostile compartment.

Cathepsin S is a cysteine protease that has been implicated in inflammatory bowel diseases (IBD) for its ability to promote visceral pain. Given its pro-inflammatory roles, we hypothesized that cathepsin S would drive other symptoms associated with IBD. Using activity-based probes, we investigated cysteine cathepsin activation in human and murine colitis. We observed a significant increase in fecal cathepsin S in patients with ulcerative colitis compared to healthy controls, while cathepsin S in mucosal biopsies was unchanged. Mice with experimental colitis exhibited a modest increase in mucosal activity of both cathepsin S and X compared to naïve mice. Luminal secretion of cathepsin S was dramatically increased upon colitis induction, although differences between mouse colonies were observed. To investigate the contribution of cathepsin S and cathepsin X to colitis, we induced colitis in cathepsin-deficient mice. Cathepsin X-deficient mice exhibited no clear differences in disease indicators compared to wild-type mice. While cathepsin S-deficient mice exhibited less rectal bleeding, less splenomegaly and marginally improved histological scores, weight loss, diarrhea, colon shortening, and myeloperoxidase activity were not significantly different from wild-type mice. To determine whether pharmacologic inhibition of cathepsin S activity would ameliorate symptoms of colitis, a reversible inhibitor LY3000328 was administered to mice at the initiation of colitis. LY3000328 provoked a clear upregulation of cathepsin S and L activity in the mucosa, most likely through a compensatory mechanism. This increase in protease activity was associated with exacerbated histological scores and slight splenomegaly. Collectively, these results suggest that cathepsin S, but not cathepsin X, may contribute to some of the symptoms of experimental colitis. While cathepsin S has potential to be a therapeutic target in colitis, improved strategies to sustain its inhibition are required in future.

Coiled-coil domain-containing protein 41 (CCDC41), a core component of centriolar distal appendages involved in centriole assembly and ciliary vesicle docking, has remained functionally uncharacterized in oocyte meiosis. Here, it is demonstrated that CCDC41 is a versatile player in three key steps of oocyte meiosis. First, CCDC41 depletion significantly impaired meiotic resumption, a defect mechanistically linked to reduced Cyclin B1 accumulation, compromised cyclin-dependent kinase 1 (CDK1) activation, and dysregulation of the anaphase-promoting complex/cyclosome (APC/C) co-activators Cdc20 homolog 1 (CDH1) and cell division cycle 20 (CDC20). Notably, this arrest is rescued by pharmacological inhibition of CDC20. Second, CCDC41 knockdown disrupted spindle cortical migration, a defect ascribed to impaired fusion of RAS oncogene family (Rab11a)-positive vesicles with the plasma membrane, which in turn prevented the anchoring of cytoplasmic F-actin to the cortex. Third, CCDC41 depletion accelerated anaphase onset by prematurely silencing the spindle assembly checkpoint and enhancing CDC20-mediated Cyclin B1 degradation. Mechanistically, CCDC41 localizes to lysosomes, and its loss delays RAS oncogene family member 7 (Rab7)-positive late endosome fusion with these organelles, causing cytoplasmic dispersion of active cathepsin. Critically, pharmacological or genetic inhibition of cathepsin B restored meiotic progression in CCDC41-deficient oocytes. Collectively, the findings establish that CCDC41 is essential for faithful meiotic completion, it regulates CDC20 activity through promoting cathepsin delivery to lysosomes, and ensures proper spindle migration via Rab11a-dependent F-actin anchoring.

Dendritic cells (DCs) are among the first immune cells to detect viral invasion and play a central role in initiating and shaping antiviral immune responses. Many innate and adaptive immune functions of DCs are regulated by cathepsins, proteolytic enzymes primarily found in acidic endolysosomal compartments. Different DC subsets exhibit distinct cathepsin expression patterns, influencing their functional capacities and interactions with viruses. In DCs, cathepsins contribute to virus sensing through innate receptors, regulate cytokine production and DC migration, and are essential for viral antigen degradation and loading onto MHC molecules for T-cell activation. Many viruses, however, have evolved mechanisms to alter cathepsin expression and activity, thereby subverting DC function and promoting their own persistence. Indeed, cathepsins can facilitate viral entry into DCs, promote viral replication, and support immune evasion strategies. In this review, we summarize recent advances in understanding the role of cathepsins in DC-virus interactions, emphasizing both how DCs exploit cathepsins to generate protective immune responses and how viruses manipulate cathepsin activity to their advantage. We particularly focus on clinically relevant viral pathogens, including HIV, influenza virus, hepatitis C virus, human cytomegalovirus, Ebola virus, and SARS-CoV-2, to illustrate the multifaceted influence of cathepsins on DC biology during viral infection.

INTRODUCTIONTauopathies involve progressive accumulation of abnormal tau species that disrupt the autophagy‐lysosomal pathway (ALP), critical for degrading intracellular macromolecules and aggregates, leading to toxicity and cell death. This study examines how overexpression of the N‐terminally truncated Tau35 protein affects proteolytic pathways, including autophagy and endo‐lysosomal processes.METHODSUsing the Tau35 mouse model and SH‐SY5Y human neuroblastoma cells stably expressing Tau35 or full‐length tau, we assessed protein degradation and lysosomal function via Western blotting, proteomics of lysosome‐enriched brain fractions, cathepsin activity assays, endocytosis/proteolysis assays, and live‐cell imaging using LysoTracker.RESULTSWe identified early endo‐lysosomal alterations associated with Tau35 expression, including increased endocytosis, disrupted autophagic flux, proteolytic impairment, and lysosomal motility defects.DISCUSSIONThese findings extend previous research by elucidating Tau35‐induced dysfunction in intracellular degradation systems and offer mechanistic insight into tauopathy progression. This work provides a foundation for developing targeted therapies to restore acidification, proteostasis, and lysosomal function in tauopathies.HighlightsTau35, an N‐terminally truncated tau fragment, disrupts proteolytic pathways: We show that Tau35 overexpression leads to significant alterations in autophagy and endo‐lysosomal function.Endo‐lysosomal dysfunction is an early pathological event: Our findings demonstrate early‐stage increases in endocytosis, impaired proteolytic activity, altered autophagic flux, and disrupted lysosomal motility in Tau35‐expressing models.In vivo and in vitro models confirm consistent pathogenic signatures: Parallel studies in a Tau35 mouse model and SH‐SY5Y cells reveal converging cellular and molecular dysfunctions.Lysosome‐enriched proteomics reveals novel pathway alterations: Proteomic profiling of lysosomal fractions identifies Tau35‐specific protein dysregulation contributing to disease pathology.Mechanistic insights into tauopathy progression: These results provide a mechanistic understanding of how truncated tau species contribute to neuronal dysfunction, offering a rationale for targeting endo‐lysosomal pathways in therapeutic development.

The immune system is increasingly recognized as playing an important role in the pathogenesis of the neurodegenerative movement disorder Parkinson’s disease (PD). In particular, key PD risk genes LRRK2 (which encodes leucine-rich repeat kinase 2) and GBA1 (which encodes glucocerebrosidase) are highly expressed in monocytes and implicated in the regulation of immune inflammatory pathways and lysosomal function. While preclinical studies demonstrate that missense mutations in LRRK2 and GBA1 can modulate the response to inflammatory stimuli, studies in primary immune cells from PD patients harboring these mutations are few. Therefore, peripheral blood mononuclear cells were obtained from idiopathic PD patients, (n = 42), as well as PD patients with GBA1 (n = 15) and LRRK2 (n = 13) mutations, and neurologically normal controls (n = 36). Cells were stimulated with interferon gamma, which strongly induces expression of the LRRK2 protein, and treated with and without the LRRK2 kinase inhibitor MLi-2. Live and fixed cell flow cytometry panels were used to measure the activities of LRRK2 and glucocerebrosidase, as well as cathepsin activity and the expression of the human leukocyte antigen receptor (HLA-DR) in classical, intermediate, and non-classical monocytes. Interferon gamma stimulation had marked effects on LRRK2 levels and phosphorylation of the LRRK2 substrate Rab10, as well as effects on the expression of HLA-DR and cathepsin activity, with some mutation-specific and monocyte-type-specific outcomes. These results help to advance understanding of how risk genes may interact with immune stimuli in the context of PD.

Joint inflammation is a hallmark of post-traumatic osteoarthritis (PTOA) progression and a recognized driver of articular destruction and symptoms. Despite its known pathological role, inflammation has not been successfully targeted to treat PTOA. With the hypothesis that blocking the acute influx of systemically-derived immune cells can mitigate injury-induced inflammation and downstream PTOA disease severity, we targeted immune cell recruitment via systemically-administered poly salicylic acid (PolySA) particles. This formulation targets immune cells in circulation, namely neutrophils and monocytes, to inhibit their vascular extravasation into injured tissue. Employing a murine joint injury model, we show that PolySA particles reduced neutrophil and monocyte recruitment to the synovium by >50% when administered acutely after injury. Sex-specific therapeutic effects of PolySA emerged 7d post-ACLR, whereby female knee joints exhibited increased cathepsin activity and alleviation of knee hyperalgesia. Despite also observing reduced immune cell recruitment in male mice treated with PolySA, therapeutic effects were entirely absent in males. RNAseq of female synovium revealed a transcriptomic signature indicative of accelerated immune resolution and matrix remodeling in PolySA-treated female mice. Analyses at a timepoint of established disease showed that PolySA-treated female mice exhibited sustained pain alleviation, reduced osteophyte formation, and decreased histopathological PTOA and synovitis severity scores. Together, these findings indicate that blocking acutely-recruited immune cells to the local joint microenvironment via systemic PolySA particle treatment is a promising therapeutic for PTOA prevention by reprogramming early injury-induced inflammation.

Fine segmentation-driven quality enhancement of unrinsed surimi: Decoupling component differentials for surimi performance.

Cellular toxicity induced by β-Amyloid (Aβ) peptide involves lamin A and lamin B fragmentation (LF),and inhibiting LF mitigates cytotoxicity; therefore, we aimed to identify novel inhibitors of Aβ-induced LF. We identified Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) and Nα-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) as inhibitors that reduce Aβ42-induced LF, alter nuclear morphology, and activate apoptotic caspases in Aβ42-treated cells. TLCK demonstrated a suppressive effect on Aβ42-induced cell death, whereas TPCK, which is inherently cytotoxic, did not. Both TLCK and TPCK decreased the activation and activity of cathepsin L (CL) which known to be implicated in catalyzing Aβ42-induced LF. TLCK and TPCK have a suppressive effect on Aβ42-induced LF in which CL is implicated. Our findings will contribute to the further investigation of Aβ-related pathology and cell death induced by other agents inhibited by TLCK and TPCK. While our findings highlight potential mechanisms underlying AD-related pathology in HeLa and SH-SY5Y cells but future validation in primary neurons, astrocytes, or animal models will be necessary to confirm these observations.

Abstract Background and Aims Functional warm ischaemia (fWIT)—the period after the donors’ blood pressure drops below 50 mmHg following withdrawal of life support until organ cold perfusion—is an inherent part of the donation after circulatory death (DCD) pathway. Causing organ hypoperfusion and cellular energy depletion, fWIT triggers a cascade of ischaemic injury, which is associated with increased incidence of delayed graft function following DCD kidney transplant. Ischaemia causes metabolic dysregulation and disruption to cellular homeostasis, which induces morphological changes and protease dysregulation, contributing to kidney injury. However, the mechanisms of injury associated with fWIT are yet to be described. To address this gap, this study used specialised proteomic techniques coupled with bioinformatics to investigate changes in endogenous peptide abundance and protease activity associated with prolonged fWIT. Method DCD kidney biopsies (n = 42) with short (<25 minutes) and long fWIT (30–107 minutes), obtained from the QUality in Organ Donation (QUOD) biobank were analysed using High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) methods combined with bioinformatic methods. Degradation profiles and changes in protease abundance were further quantified using Western blotting, in both kidney tissue and immortalised podocytes and proximal tubule epithelial cells (PTECs) cultured under hypoxic and varying glucose conditions. Results Mass spectrometry analysis identified 2,563 endogenous peptides which were mapped to 1,022 distinct proteins. Of these, seven endogenous peptides—associated with proximal tubule injury, metabolic pathways, and the cytoskeleton—showed increased abundance as fWIT was prolonged (Fig. 1A). Pathway analysis of the mapped proteins revealed significant dysregulation in metabolic processes, particularly affecting glucose, lipid and vitamin metabolism. Notably, the rate-limiting gluconeogenesis enzyme, fructose-1,6-bisphosphatase (FBP1), exhibited significant degradation with prolonged fWIT (Fragment 1 and Fragment 2; P = 0.0004, FDR 5%). Western blot analysis confirmed this result, showing marked increased in FBP1 degradation as fWIT increased (P = 0.0003) (Fig. 1B). Protease activity was also significantly dysregulated as fWIT was prolonged (Fig. 1C). Legumain activity increased (P = 0.00000009, FDR 5%) with fWIT, yet the activity of Cathepsin B—a substrate of Legumain—decreased (P = 0.004, FDR 5%). Interestingly, Legumain abundance, measured by western blot, did not change with fWIT, yet was significantly reduced in glucose-starved PTECs (but not podocytes), indicating a cell-type-specific metabolic disruption associated with glucose availability and hypoxia. Cathepsin B abundance decreased in kidney biopsies as fWIT increased (P = 0.03). In hypoxic PTECs and podocytes, Cathepsin B abundance decreased after 6 h and 24 h, although this change did not reach statistical significance. Conclusion Prolonged fWIT induces metabolic and protease dysregulation in DCD kidneys, which may impair posttransplant function. As ischemia time increases, disruption of glucose metabolism likely drives changes in protease activity and subsequent protein degradation. Legumain is a high specificity protease that along with its regulatory components emerge as promising therapeutic targets to mitigate injury in DCD kidneys and potentially improve transplant outcomes.

Polystyrene microplastic (PS-MP), known as a white pollutant, exhibited adverse effects on aquatic and terrestrial animals. The present study aims to evaluate the dose-dependent effect of polystyrene microplastics on skeletal muscle energy metabolism in Wistar rats. PS-MP was administered orally in Wistar rats at doses of 0.5mg/L, 5mg/L, and 50mg/L in drinking water for 28 days daily. After the treatment, metabolic profile and tissue histological analyses were performed. Average food consumption by the treated rats was decreased by PS-MPs. Glycogen and pyruvate contents were depleted in a dose-responsive fashion. Lactate dehydrogenase and transaminase activities were decreased by PS-MP exposure. Free amino nitrogen was mobilized from blood to skeletal muscle in response to stress. Protein content depleted in the muscular tissue whereas enhanced carbonylated protein formation. Pronase and cathepsin activities were increased by PS-MP. Inhibited TCA cycle enzyme activities were observed in the target tissue. Moreover, muscle hypertrophy, nuclear migration, and fibrillation were seen in histological sections. Decreased food consumption by PS-MP exposure could promote glucose scarcity in blood. Depletion of muscular glycogen may result from increased glycogenolysis to replenish loss of blood glucose. Reduction in pyruvate content may result from decreased glycolysis which could perturb the lactate dehydrogenase function. Lack of transaminase in the target tissue was indicative of tissue damage. Muscular protein breakdown might be due to oxidative denaturation of native proteins as well as increased proteolysis. Due to less pyruvate production, the TCA cycle enzyme functions were suppressed. Histopathological studies established significant degenerative changes in muscular morphology following PS-MP exposure. The present study suggests that PS-MP perturbed skeleto-muscular energy metabolism and promoted muscle fiber degeneration following sub-acute exposure.

Fluorescent molecular probes have frequently been used to monitor lysosomal health, localization, abundance, and movement through the detection of acidic organelles and lysosomal enzyme activity. Flow cytometry technology provides rapid and accurate analysis of single cells (neurons) or particles (lysosomes) in suspension through laser detection. Herein, we describe how to detect lysosomes via LysoTracker™ and Magic Red® Cathepsin Activity assays in iPSC-derived human neuron cultures by flow cytometry.

Musculoskeletal degenerative diseases significantly compromise patient quality of life through muscle loss, bone fragility, and cartilage degeneration. Given the established link between vascular aging and musculoskeletal disorders, the vascular-skeletomuscular axis represents a complex network wherein conditions interact via mechanisms including impaired blood flow, inflammation, oxidative stress, and hormonal imbalances. As pharmacological interventions targeting lysosomal cathepsins face developmental and application challenges, exploring non-pharmacological alternatives becomes imperative. This review investigates the regulatory roles of the lysosomal cathepsin family, especially cathepsins B, K, and L, in musculoskeletal degenerative diseases and evaluates how exercise interventions modulate their activities. It proposes exercise as a viable non-pharmacological strategy to selectively influence lysosomal cathepsin functions, thereby offering therapeutic potential in managing vascular-musculoskeletal degenerative diseases. The review elucidates several key concepts: the intricate interaction between vascular aging and musculoskeletal degenerative pathologies; the pivotal roles of lysosomal cathepsins in disease progression through metabolic regulation, inflammatory modulation, macrophage polarization, and autophagy dysfunction; and the multifaceted mechanisms by which exercise influences cathepsin activity, including calcium homeostasis, cellular energy metabolism enhancement, inflammatory response reduction, autophagy stimulation, myokine secretion, and vascular function improvement. This comprehensive analysis provides novel insights into exercise as a selective modulator of lysosomal cathepsin activity, highlighting its significant potential for advancing diagnostic and clinical therapeutic approaches.

BackgroundEarlier research studies employing observational methods have suggested a possible relationship between the activity of cathepsin Z and thyroid cancer (TC). However, the causal relationship linking the cathepsin Z to TC has yet to be fully established, especially for different subtypes of TC.MethodsThe study employed accessible genomewide association study (GWAS) datasets to conduct bidirectional Mendelian randomization (MR) analyses. The primary approach for conducting MR analysis was the application of inverse variance weighting (IVW).ResultsThe MR analysis indicated that elevated cathepsin Z levels are positively linked to an elevated risk of papillary TC (PTC) development. In contrast, reverse MR indicated that PTC cannot contribute to increasing cathepsin Z levels.ConclusionOur MR analysis suggests a causal role of cathepsin Z in the development of PTC, offering valuable insights for future mechanistic studies and potential clinical applications targeting cathepsin-mediated pathways in cancer.