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Cathepsin D (CTSD) is a lysosomal aspartic protease and its inherited deficiency causes a severe pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10. The lysosomal dysfunction in the affected patients leads to accumulation of undigested lysosomal cargo especially in none-dividing cells, such as neurons, resulting in death shortly after birth. To explore which proteins are mainly affected by the lysosomal dysfunction due to CTSD deficiency, Lund human mesencephalic (LUHMES) cells, capable of inducible dopaminergic neuronal differentiation, were treated with Pepstatin A. This inhibitor of "acidic" aspartic proteases caused accumulation of acidic intracellular vesicles in differentiating LUHMES cells. Pulse-chase experiments involving stable isotope labelling with amino acids in cell culture (SILAC) with subsequent mass-spectrometric protein identification and quantification were performed. By this approach, we studied the degradation and synthesis rates of 695 and 680 proteins during early and late neuronal LUHMES differentiation, respectively. Interestingly, lysosomal bulk proteolysis was not altered upon Pepstatin A treatment. Instead, the protease inhibitor selectively changed the turnover of individual proteins. Especially proteins belonging to the mitochondrial energy supply system were differentially degraded during early and late neuronal differentiation indicating a high energy demand as well as stress level in LUHMES cells treated with Pepstatin A., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background and Objectives: Fibrosis contributes to 45% of deaths in industrialized nations and is characterized by an abnormal accumulation of extracellular matrix (ECM). There are no specific anti-fibrotic treatments for liver fibrosis, and previous unsuccessful attempts at drug development have focused on preventing ECM deposition. Because liver fibrosis is largely acknowledged to be reversible, regulating fibrosis resolution could offer novel therapeutical options. However, little is known about the mechanisms controlling ECM remodeling during resolution. Changes in proteolytic activity are essential for ECM homeostasis and macrophages are an important source of proteases. Herein, in this study we evaluate the role of macrophage-derived cathepsin D (CtsD) during liver fibrosis., Methods: CtsD expression and associated pathways were characterized in single-cell RNA sequencing and transcriptomic datasets in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD and hepatocyte-CtsD knock-out mice., Results: Analysis of single-cell RNA sequencing datasets demonstrated CtsD was expressed in macrophages and hepatocytes in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD (CtsD ΔMyel ) and hepatocyte-CtsD knock-out mice. CtsD deletion in macrophages, but not in hepatocytes, resulted in enhanced liver fibrosis. Both inflammatory and matrisome proteomic signatures were enriched in fibrotic CtsD ΔMyel livers. Besides, CtsD ΔMyel liver macrophages displayed functional, phenotypical and secretomic changes, which resulted in a degradomic phenotypical shift, responsible for the defective proteolytic processing of collagen I in vitro and impaired collagen remodeling during fibrosis resolution in vivo. Finally, CtsD-expressing mononuclear phagocytes of cirrhotic human livers were enriched in lysosomal and ECM degradative signaling pathways., Conclusions: Our work describes for the first-time CtsD-driven lysosomal activity as a central hub for restorative macrophage function during fibrosis resolution and opens new avenues to explore their degradome landscape to inform drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Prodiginines have been studied extensively for their anticancer activity, however, the majority of the research has focused on prodigiosin. In this study, cycloheptylprodigiosin (S-1) is extracted from marine bacterium Spartinivicinus ruber MCCC 1K03745 T , and its anticancer property was investigated. It exhibits remarkable cytotoxicity against a panel of human lung cancer cell lines, with the IC 50 values ranging from 84.89 nM to 661.2 nM. After 6 h of treatment, S-1 gradually accumulates on mitochondria and lysosomes. While lower doses of S-1 induce cell cycle arrest, treatment with higher doses results in cell death in apoptotic independent manner in both NCI-H1299 and NCI-H460 cell lines. Interestingly, treatment with S-1 leads to the accumulation of LC3B-II via pathways that vary among different cell lines. In addition to its role as an autophagy inhibitor, S-1 also promotes autophagy initiation as demonstrated by the increment of EGFP fragment in the EGFP-LC3 degradation assay, however, inhibition of autophagy does not rescue cells from death induced by S-1. Mechanistically, S-1 impairs autophagic flux through disrupting acidic lysosomal pH and blocking the maturation of cathepsin D. Moreover, treatment with S-1 enhanced secretion of both pro- and mature forms of cathepsin D, coincident with disintegration of trans-Golgi network. Interestingly, S-1 does not induce ferroptosis, pyroptosis or necroptosis in NCI-H1299 cells. However, treatment of NCI-H460 cells with S-1 induces methuosis, which can be suppressed by Rac1 inhibitor EHT 1864. Our data demonstrate that S-1 is an effective anticancer agent with potential therapeutic application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. There are conflicting results regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. This study aimed to evaluate the expression and prognostic significance of Cathepsin D in early-stage breast cancer., Methods: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n = 954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of Cathepsin D expression with clinicopathological parameters and prognosis was evaluated., Results: Cathepsin D expression was positive in 71.2% (679/954) of breast cancer tumours. Positive expression of Cathepsin D was significantly associated with high histological grade (p = 0.007), pleomorphism (p = 0.002), poor Nottingham Prognostic Index (NPI) score (p < 0.002), recurrence (p = 0.005) and distant metastasis (p < 0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer-specific survival (p = 0.001), higher risk of recurrence (p = 0.001) and distant metastasis (p < 0.0001). ER-positive tumours expressing Cathepsin D and treated with tamoxifen demonstrated a significantly higher risk of distant metastasis., Conclusion: Cathepsin D expression significantly predicts poor prognosis in breast cancer and is associated with variables of poor prognosis and shorter outcome. The strong association of Cathepsin D with aggressive tumour characteristics and poor outcomes warrants further research of its potential as a therapeutic target The results also suggest a possible interaction between Cathepsin D and tamoxifen therapy in ER-positive breast cancer which needs further investigation to elucidate the underlying mechanisms., (© 2024. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is the most prevalent and lethal class of severe motor neuron diseases (MND) with no efficacious treatment. The pathogenic mechanisms underlying ALS remain unclear. Nearly 90% of patients exhibit sporadic onset (sALS). Therefore, elucidating the pathophysiology of ALS is imperative. Long non-coding RNA (lncRNA) is a large class of non-coding RNAs that regulate transcription, translation, and post-translational processes. LncRNAs contribute to the pathogenesis of diverse neurodegenerative disorders and hold promise as targets for interference in the realm of neurodegeneration. However, the mechanisms of which lncRNAs are involved in ALS have not been thoroughly investigated. We identified and validated a downregulated lncRNA, lnc-HIBADH-4, in ALS which correlated with disease severity and overall survival. Lnc-HIBADH-4 acted as a "molecular sponge" regulating lysosomal function through the lnc-HIBADH-4/miR-326/CTSD pathway, thereby impacting autophagy-lysosome dynamics and the levels of cell proliferation and apoptosis. Therefore, this study discovered and revealed the role of lnc-HIBADH-4 in the pathogenesis of ALS. With further research, lnc-HIBADH-4 is expected to provide a new biomarker in the diagnosis and treatment of ALS., (© 2023. The Author(s).)

Objective: 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)., Methods: 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., Results: 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., Interpretation: 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., (© 2024 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synuclein Ser129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synuclein Ser129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4 -/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4 -/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her's dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies., (Copyright © 2024 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aβ) peptides. The extracellular deposition of Aβ peptides in human AD brain causes neuronal death. Therefore, it has been found that Aβ peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD., (© 2024. The Author(s).)

Colorectal cancer (CRC) is a leading cause of death worldwide. Conventional therapies are available with varying effectiveness. Acetate, a short-chain fatty acid produced by human intestinal bacteria, triggers mitochondria-mediated apoptosis preferentially in CRC but not in normal colonocytes, which has spurred an interest in its use for CRC prevention/therapy. We previously uncovered that acetate-induced mitochondrial-mediated apoptosis in CRC cells is significantly enhanced by the inhibition of the lysosomal protease cathepsin D (CatD), which indicates both mitochondria and the lysosome are involved in the regulation of acetate-induced apoptosis. Herein, we sought to determine whether mitochondrial function affects CatD apoptotic function. We found that enhancement of acetate-induced apoptosis by CatD inhibition depends on oligomycin A-sensitive respiration. Mechanistically, the potentiating effect is associated with an increase in cellular and mitochondrial superoxide anion accumulation and mitochondrial mass. Our results provide novel clues into the regulation of CatD function and the effect of tumor heterogeneity in the outcome of combined treatment using acetate and CatD inhibitors.

Background: Microvascular complications are the major outcome of type 2 diabetes progression, and the underlying mechanism remains to be determined., Methods: High-throughput RNA sequencing was performed using human monocyte samples from controls and diabetes. The transgenic mice expressing human CTSD (cathepsin D) in the monocytes was constructed using CD68 promoter. In vivo 2-photon imaging, behavioral tests, immunofluorescence, transmission electron microscopy, Western blot analysis, vascular leakage assay, and single-cell RNA sequencing were performed to clarify the phenotype and elucidate the molecular mechanism., Results: Monocytes expressed high-level CTSD in patients with type 2 diabetes. The transgenic mice expressing human CTSD in the monocytes showed increased brain microvascular permeability resembling the diabetic microvascular phenotype, accompanied by cognitive deficit. Mechanistically, the monocytes release nonenzymatic pro-CTSD to upregulate caveolin expression in brain endothelium triggering caveolae-mediated transcytosis, without affecting the paracellular route of brain microvasculature. The circulating pro-CTSD activated the caveolae-mediated transcytosis in brain endothelial cells via its binding with low-density LRP1 (lipoprotein receptor-related protein 1). Importantly, genetic ablation of CTSD in the monocytes exhibited a protective effect against the diabetes-enhanced brain microvascular transcytosis and the diabetes-induced cognitive impairment., Conclusions: These findings uncover the novel role of circulatory pro-CTSD from monocytes in the pathogenesis of cerebral microvascular lesions in diabetes. The circulatory pro-CTSD is a potential target for the intervention of microvascular complications in diabetes., Competing Interests: Disclosures None.

M2-like tumor-associated macrophages (TAMs) are risk factors for cancer progression and metastasis. However, the mechanisms underlying their polarization are still not fully understood. Although cathepsin D (Cat D) has been reported as a procarcinogenic factor, little is known about the functional role of Cat D in the tumor microenvironment (TME). This study aimed to explore the effect and molecular mechanisms of Cat D in the TME. Cat D knockout (KO) altered the cytokine secretion pattern and induced TAM reprogramming from the M2 to M1 subtype, thereby preventing epithelial-mesenchymal transition and tumor metastasis. Mechanistically, we identified transforming growth factor beta-induced protein (TGFBI) as a Cat D target protein that is specifically associated with TAM polarization. Elevated TGFBI expression in Cat D KO cancer cells resulted in a decline in M2-like TAM polarization. Our RNA-sequencing results indicated that the cancer cell-secreted chemokine CCL20 is a major secretory chemokine for Cat D-TGFBI-mediated TAM polarization. In contrast, Cat D overexpression accelerated TAM polarization into M2-like cells by suppressing TGFBI expression. In addition, the double Cat D and TGFBI KO rescued the inhibitory effects of Cat D KO on tumor metastasis by controlling TAM and T-cell activation. These findings indicated that Cat D contributes to cancer metastasis through TGFBI-mediated TAM reprogramming. Cat D deletion inhibits M2-like TAM polarization through TGFBI-mediated CCL20 expression, reprogramming the immunosuppressive TME. Our results open a potential new avenue for therapy focused on eliminating tumor metastasis., (© 2024. The Author(s).)

Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, "naive" progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-β-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and β-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-β-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-β-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling., (© 2023. The Author(s).)

Cathepsin D (CTSD) is an aspartic endopeptidase, however, we found that it was also capable of enzymatic digestion of nucleic acids (NAs). The purpose of this study was to investigate the basic properties of CTSD enzymatic activity on NAs, and explore the degradation mechanism. The results showed that NAs were efficiently digested between pH 3.0 and 5.0, and the optimum pH was 3.5. CTSD exhibited optimum activity at the temperature of 50°C. The degradation rate was improved with an increased CTSD concentration, and NAs were digested to an enzyme concentration of 0.001%, at which point, NAs were no longer digested. Ca 2+ and Mg 2+ at low concentrations of 5 mM promoted the digestion remarkably. As the protein substrate for CTSD, both Hb and BSA had no effect on DNA degradation, even when the molar ratio of protein:DNA was 10 4 :1. Kinetic parameters of K m and k cat /K m value were (42 ± 1) μM and (1.62 ± 0.1) × 10 -2 s -1 mM -1 respectively, using real-time quantitative PCR (RT-PCR). Specially, pepstatin A which is the specific aspartic protease inhibitor exhibited inhibitory effect on NA digestion by CTSD as well, suggesting that the catalytic active site of CTSD for NAs might be the same as protein. A brief degradation mechanism is discussed. The present study may change the cognition of CTSD specificity for substrate and contribute greatly to enzymology of CTSD., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Aims: To investigate the pathogenesis of a disease in takahē (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons., Methods: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy., Results: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other., Conclusions: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity that is extensively expressed in the lung, pancreas, thymus, kidney, liver, skin, and brain. Owing to its specific enzymatic activity, CatH has critical effects on the regulation of biological behaviours of cancer cells and pathological processes in brain diseases. Moreover, a neutral pH level is optimal for CatH activity, so it is expected to be active in the extra-lysosomal and extracellular space. In the present review, we describe the expression, maturation, and enzymatic properties of CatH, and summarize the available experimental evidence that mechanistically links CatH to various physiological and pathological processes. Finally, we discuss the challenges and potentials of CatH inhibitors in CatH-induced disease therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cathepsin D (CTSD) is a major lysosomal protease harboring an N-terminal signal peptide (amino acids 1-20) to enable vesicular transport from endoplasmic reticulum to lysosomes. Here, we report the possibility of a mitochondrial targeting sequence and mitochondrial localization of CTSD in cells. Live-cell imaging analysis with C-terminal enhanced green fluorescent protein-tagged CTSD (EGFP-CTSD) indicated that CTSD localizes to mitochondria. CTSD amino acids 21-35 are responsible for its mitochondrial localization, which exhibit typical features of mitochondrial targeting sequences, and are evolutionarily conserved. A proteinase K protection assay and sucrose gradient analysis showed that a small population of endogenous CTSD molecules exists in mitochondria. These results suggest that CTSD is a dual-targeted protein that may localize in both lysosomes and mitochondria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80-90% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development, and detailed characterization of a dual-function "TRE-Lox" system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of a target gene-the lysosomal aspartyl protease cathepsin D (CatD)-based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5' end of the endogenous CatD gene ( CTSD ). Using an optimized reverse-tetracycline transrepressor (rtTR) variant fused with the Krüppel-associated box (KRAB) domain, we show that CatD expression can be disrupted by as much as 98% in mouse embryonic fibroblasts (MEFs). This system is highly sensitive to Dox (IC 50 = 1.46 ng/mL) and results in rapid (t 1/2 = 0.57 d) and titratable downregulation of CatD. Notably, even near-total disruption of CatD expression was completely reversed by withdrawal of Dox. As expected, transient expression of Cre recombinase results in complete deletion of the CTSD gene. The dual functionality of this novel system will facilitate future studies of the involvement of CatD in various diseases, particularly those attributable to partial loss of CatD function. In addition, the TRE-Lox approach should be applicable to the regulation of other target genes requiring more complete disruption than can be achieved by traditional methods.

Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana , respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway.

Fluorescent cathepsin probes were prepared by modification of peptidic substrates for cathepsin B (CTSB) and cathepsin D (CTSD) with FRET pairs. Fluorophores with distinguishable emission characteristics were applied to CTSB and CTSD probes with their appropriate quenchers to simultaneously monitor the activity of CTSB and/or CTSD. Conjugation of both the CTSB and CTSD probes with short single-stranded DNA drastically increased their reactivity to cathepsins over the parent probes possibly by improving their solubility. The activity of CTSB and CTSD were simultaneously detected by using these orthogonal FRET-based cathepsin probes., (© 2022 Wiley-VCH GmbH.)

Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N -glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O -glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O -glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

L-serine is a non-essential amino acid endogenously produced by astrocytes and is abundant in human diets. Beneficial roles of the metabolic products from L-serine in various conditions in the brain including neuronal development have been reported. Through several preclinical studies, L-serine treatment was also shown to offer beneficial therapeutic effects for brain damage such as ischemic stroke, amyotrophic lateral sclerosis, and Parkinson's disease. Despite evidence for the value of L-serine in the clinic, however, its beneficial effects on the propionic acid (PPA)-induced neuronal toxicity and underlying mechanisms of L-serine-mediated neuroprotection are unknown. In this study, we observed that PPA-induced acidic stress induces abnormal lipid accumulation and functional defects in lysosomes of hippocampal neurons. L-serine treatment was able to rescue the structure and function of lysosomes in PPA-treated hippocampal neuronal cells. We further identified that L-serine suppressed the formation of lipid droplets and abnormal lipid membrane accumulations inside the lysosomes in PPA-treated hippocampal neuronal cells. Taken together, these findings indicate that L-serine can be utilized as a neuroprotective agent for the functionality of lysosomes through restoration of cathepsin D in disease conditions.

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy., (© 2022 Wiley Periodicals LLC.)

Neuronal ceroid lipofuscinosis is one of many neurodegenerative storage diseases characterized by excessive accumulation of lipofuscins. CLN10 disease, an early infantile neuronal ceroid lipofuscinosis, is associated with a gene that encodes cathepsin D (CtsD), one of the major lysosomal proteases. Whole body CtsD-knockout mice show neurodegenerative phenotypes with the accumulation of lipofuscins in the brain and also show defects in other tissues including intestinal necrosis. To clarify the precise role of CtsD in the central nervous system (CNS), we generated a CNS-specific CtsD-knockout mouse (CtsD-CKO). CtsD-CKO mice were born normally but developed seizures and their growth stunted at around postnatal day 23 ± 1. CtsD-CKO did not exhibit apparent intestinal symptoms as those observed in whole body knockout. Histologically, autofluorescent materials were detected in several areas of the CtsD-CKO mouse's brain, including: thalamus, cerebral cortex, hippocampus, and cerebellum. Expression of ubiquitin and autophagy-associated proteins was also increased, suggesting that the autophagy-lysosome system was impaired. Microglia and astrocytes were activated in the CtsD-CKO thalamus, and inducible nitric oxide synthase (iNOS), an inflammation marker, was increased in the microglia. Interestingly, deposits of proteinopathy-related proteins, phosphorylated α-synuclein, and Tau protein were also increased in the thalamus of CtsD-CKO infant mice. Considering these results, we propose thatt the CtsD-CKO mouse is a useful mouse model to investigate the contribution of cathepsin D to the early phases of neurodegenerative diseases in relation to lipofuscins, proteinopathy-related proteins and activation of microglia and astrocytes., (© 2022. The Author(s).)

Yolk processing pathways vary in the oocytes of benthophil and pelagophil teleosts. The present study investigated the yolk processing pattern in the oocytes of the fresh water catfish Clarias gariepinus at vitellogenic, maturation, and ovulated stages. This study concludes that during maturation stage, an electrophoretic shift in the major peptide band on Polyacrylamide gel electrophoresis (PAGE) occurs due to a decrease in the size of the yolk protein. The PMF spectrum of corresponding peptides from vitellogenic and ovulated oocytes revealed a difference in the minor ions. A minor difference in the molecular weight of the corresponding peptides occurs due to a difference in their amino acid composition. Maximal activity of the proteases cathepsin D and cathepsin B was observed in the vitellogenic oocytes, thus confirming their role in the processing of yolk. A significant transient increase in the activity of cathepsin B in the mature oocytes also suggests its role in oocyte maturation., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

We aim to investigate the mechanisms underlying the beneficial effects of exercise rehabilitation (ER) and/or astragaloside (AST) in counteracting amyloid-beta (Aβ) pathology. Aβ oligomers were microinjected into the bilateral ventricles to induce Aβ neuropathology in rats. Neurobehavioral functions were evaluated. Cortical and hippocampal expressions of both BDNF/TrkB and cathepsin D were determined by the western blotting method. The rat primary cultured cortical neurons were incubated with BDNF and/or AST and ANA12 followed by exposure to aggregated Aβ for 24 h. In vivo results showed that ER and/or AST reversed neurobehavioral disorders, downregulation of cortical and hippocampal expression of both BDNF/TrkB and cathepsin D, neural pathology, Aβ accumulation, and altered microglial polarization caused by Aβ. In vitro studies also confirmed that topical application of BDNF and/or AST reversed the Aβ-induced cytotoxicity, apoptosis, mitochondrial distress, and synaptotoxicity and decreased expression of p-TrkB, p-Akt, p-GSK3β, and β-catenin in rat cortical neurons. The beneficial effects of combined ER (or BDNF) and AST therapy in vivo and in vitro were superior to ER (or BDNF) or AST alone. Furthermore, we observed that any gains from ER (or BDNF) and/or AST could be significantly eliminated by ANA-12, a potent BDNF/TrkB antagonist. These results indicate that whereas ER (or BDNF) and/or AST attenuate Aβ pathology by reversing BDNF/TrkB signaling deficits and mitochondrial dysfunction, combining these two potentiates each other's therapeutic effects. In particular, AST can be an alternative therapy to replace ER., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Adenosine A 2A receptor (A 2A R)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A 2A R targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A 2A R and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A 2A R-interacting partners have in macrophages. Here, we aimed to identify A 2A R-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A 2A R as the "bait" and a macrophage expression library as the "prey." We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A 2A R-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A 2A R is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A 2A R in macrophages. Conversely, we demonstrate that A 2A R activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A 2A R-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroblastoma is a malignant extracranial solid tumor arising from the sympathoadrenal lineage of the neural crest and is often associated with N-MYC amplification. Cathepsin D has been associated with chemoresistance in N-MYC-overexpressing neuroblastomas. Increased EGFR expression also has been associated with the aggressive behavior of neuroblastomas. This work aimed to understand the mechanisms linking EGFR stimulation and cathepsin D expression with neuroblastoma progression and prognosis. Gene correlation analysis in pediatric neuroblastoma patients revealed that individuals bearing a high EGFR transcript level have a good prognosis only when CTSD (the gene coding for the lysosomal protease Cathepsin D, CD) is highly expressed. Low CTSD expression was associated with poor clinical outcome. CTSD expression was negatively correlated with CCNB2 , CCNA2 , CDK1 and CDK6 genes involved in cell cycle division. We investigated the biochemical pathways downstream to EGFR stimulation in human SH-SY5Y neuroblastoma cells engineered for overexpressing or silencing of CD expression. Cathepsin D overexpression decreased the proliferative potential of neuroblastoma cells through downregulation of the pro-oncogenic MAPK signaling pathway. EGFR stimulation downregulated cathepsin D expression, thus favoring cell cycle division. Our data suggest that chemotherapeutics that inhibit the EGFR pathway, along with stimulators of cathepsin D synthesis and activity, could benefit neuroblastoma prognosis.

Objective: To investigate the role of high-sugar high-fat treatment in inducing autophagy of rat retinal microvascular endothelial cells., Methods: The optimal concentrations and time points of glucose and oxidized low-density lipoprotein (ox-LDL) in inducing rat retinal microvascular endothelial cells were determined by examining the proliferate rate by CCK-8 assay. They were divided into control group (blank control), model group (treatment of 50 mM glucose and 10 μg/ml ox-LDL for 24 h), chloroquine group (treatment of 20 μM chloroquine, 50 mM glucose and 10 μg/ml ox-LDL for 24 h), resveratrol group (treatment of 50 μM resveratrol, 50 mM glucose and 10 μg/ml ox-LDL for 24 h) and MITO-Tempol group (treatment of 20 μM MITO-Tempol, 50 mM glucose and 10 μg/ml ox-LDL for 24 h). Reactive oxygen species (ROS) level in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment was measured by flow cytometry. In addition, protein levels of cathepsin B and cathepsin D in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment were examined by immunofluorescence, and protein levels of LC3 A/B and the autophagy substrate P62 were detected by Western blot., Results: Primary retinal microvascular endothelial cells were isolated from neonatal Sprague-Dawley (SD) rats. ROS level was significantly higher in model group than that of control group (P < 0.05). Compared with that of model group, ROS level was significantly reduced in chloroquine group and MITO-Tempol group, which was significantly elevated in resveratrol group (P < 0.05). Positive expressions of cathepsin B and cathepsin D were significantly reduced in model group than those of control group (P < 0.05). They were significantly elevated in chloroquine group and MITO-Tempol group, and reduced in resveratrol group than those of model group (P < 0.05). LC3 A/B and P62 were significantly upregulated in model group than those of control group (P < 0.05). Compared with those of model group, LC3 A/B and P62 were significantly downregulated in chloroquine group and MITO-Tempol group, and upregulated in resveratrol group (P < 0.05)., Conclusion: High-sugar high-fat treatment induces autophagy of rat retinal microvascular endothelial cells, which can be intervened to a certain extent by chloroquine and MITO-Tempol., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. The study design was approved by the appropriate ethics review board. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2022. Published by Elsevier Inc.)

Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623&#95;His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi -p.Gly623&#95;Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT- TGFBI and mut- TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD. Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type.

Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cathepsin D (Cath D) has been evidenced as a potential target for cancer therapy. Our previous studies revealed that TB-9, a tasiamide B derivative, exhibited highly potent inhibition against Cath D with satisfactory selectivity over Cath E and BACE1. But this compound was inactive on cell level possibly due to poor membrane permeability. Herein, we report the design, synthesis, and evaluation of two novel Cath D inhibitors (2 and 3) which combining tasiamide B scaffold with a cell penetrating peptide (CPP) specifically targeting the endolysosomal compartment. The results revealed that 2 and 3 not only retained highly potent inhibition against Cath D, but also were active against MDA-MB-231 cell lines., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aspartic proteinases are one of the four families of proteinase enzymes that are widely present in living organisms. They are involved in various physiological events, such as protein degradation, development, and host defense. However, the characterization and functional roles of aspartic proteinases remain to be elucidated in crustaceans. Here, we characterized a fragment of cathepsin D-like cDNA from red swamp crayfish, Procambarus clarkii ( Pc-cathepsin D-like ). The open reading frame of the Pc-cathepsin D-like gene contained 1152 bp, encoding a protein of 383 amino acid residues. We also evaluated the immunological role of the Pc-cathepsin D-like gene in vivo. Spatial distribution analysis revealed that the Pc-cathepsin D-like mRNA was high in the hepatopancreas, followed by the gut, gills, and hemocytes of P. clarkii . The expression levels of the Pc-cathepsin D-like gene increased following challenge with viral (polyinosinic: polycytidylic acid) and bacterial (lipopolysaccharides, peptidoglycan) PAMPs compared with PBS injection. The suppression of the Pc-cathepsin D-like gene by RNA interference significantly increased the expression of immune-associated genes. These results showed that the Pc-cathepsin D-like gene has an essential biological role in innate immune responses because it regulates the expression of immune-associated genes.

Background: Cathepsin D is a lysosomal aspartic protease encoded by the CTSD gene. It plays important roles in many biological processes. Biallelic loss-of-function mutation of CTSD is considered a cause of CLN10 disease. CLN10 is a rare autosomal recessive disorder that is one of 14 types of neuronal ceroid lipofuscinoses (NCLs). To date, only a few cases of CLN10 and 12 disease-causing mutations have been reported worldwide., Methods: Exome sequencing was performed on a 15-year-old girl with pervasive brain developmental disorder. The effects of the identified variants were investigated through multiple functional experiments., Results: There were no differences in mRNA and protein expression, intracellular localization, maturation, and proteolytic activity between the cells with the mutant CTSD gene and those with the wild-type CTSD gene., Conclusion: These results suggest that the c.863A>G (p.Glu288Gly) homozygous variant is not a pathogenic variation, but a benign variant., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

As components of the human diet with potential health benefits, flavonols are the subject of numerous studies, confirming their antioxidant, free radical scavenging and anti-inflammatory activity. Taking into consideration the postulated pathogenesis of certain CNS dysfunctions characterized by neuronal degradation, flavonols may prevent the decay of neurons in multiple pathways. Leaves of Maesa membranacea yielded several flavonol glycosides including α-rhamnoisorobin (kaempferol 7- O- α-rhamnoside) and kaempferitrin (kaempferol 3,7-di- O -α-rhamnoside). The latter compound was a major constituent of the investigated plant material. Neuroprotective effects of kaempferitrin and α-rhamnoisorobin were tested in vitro using H 2 O 2 -, 6-OHDA- and doxorubicin-induced models of SH-SY5Y cell damage. Both undifferentiated and differentiated neuroblastoma cells were used in the experiments. α-Rhamnoisorobin at a concentration range of 1-10 µM demonstrated cytoprotective effects against H 2 O 2 -induced cell damage. The compound (at 1-10 µM) was also effective in attenuating 6-OHDA-induced neurotoxicity. In both H 2 O 2 - and 6-OHDA-induced cell damage, kaempferitrin, similar to isoquercitrin, demonstrated neuroprotective activity at the highest of the tested concentrations (50 µM). The tested flavonols were not effective in counteracting doxorubicin-induced cytotoxicity. Their caspase-3- and cathepsin D-inhibitory activities appeared to be structure dependent. Inhibition of the PI3-K/Akt pathway abolished the neuroprotective effect of the investigated flavonols.

Degradation and clearance of cellular waste in the autophagic and endo-lysosomal systems is important for normal physiology and prevention of common late-onset diseases such as Alzheimer's disease (AD). Phosphatidylinostol-binding clathrin assembly protein (PICALM) is a robust AD risk factor gene and encodes an endosomal protein clathrin-binding cytosolic protein, reduction of which is known to exacerbate tauopathy. Although PICALM is known to regulate initiation of autophagy, its role in maturation of lysosomal enzymes required for proteolysis has not been studied. We sought to determine the importance of PICALM for cellular degradative function by disrupting exon 1 of PICALM using CRISPR/Cas9 in HeLa cells. PICALM disruption increased numbers of early endosomes. Proteomic analysis of endosome-enriched samples showed that disrupting exon 1 of PICALM increased the abundance of lysosomal enzymes in these organelles, and western blotting revealed disruption to processing and maturation of the lysosomal protease, cathepsin D, and a deficit in autophagy. This study shows PICALM is important for the correct maturation of lysosomal enzymes and efficient proteolytic function in the lysosome., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Timothy John Sargeant reports financial support was provided by Rebecca L Cooper Medical Research Foundation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Disruption of the mannose 6-phosphate (M-6-P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc-1-phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB -/- HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M-6-P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc-1-phosphotransferase show a fivefold increase in cathepsin D mRNA expression over control cells, accounting for the increase in cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M-6-P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB -/- HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Stroke is a leading cause of death and disability. The pathophysiological mechanisms associated with stroke are very complex and not fully understood. Lysosomal function has a vital physiological function in the maintenance of cellular homeostasis. In neurons, CTSD (cathepsin D) is an essential protease involved in the regulation of proteolytic activity of the lysosomes. Loss of CTSD leads to lysosomal dysfunction and accumulation of different cellular proteins implicated in neurodegenerative diseases. In cerebral ischemia, the role of CTSD and lysosomal function is not clearly defined. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons and the middle cerebral artery occlusion (MCAO) model of stroke to assess the role of CTSD in stroke pathophysiology. Our results show a time-dependent decrease in CTSD protein levels and activity in the mouse brain after stroke and neurons following OGD, with concurrent defects in lysosomal function. We found that shRNA-mediated knockdown of CTSD in neurons is sufficient to cause lysosomal dysfunction. CTSD knockdown further aggravates lysosomal dysfunction and cell death in OGD-exposed neurons. Restoration of CTSD protein levels via lentiviral transduction increases CTSD activity in neurons and, thus, renders resistance to OGD-mediated defects in lysosomal function and cell death. This study indicates that CTSD-dependent lysosomal function is critical for maintaining neuronal survival in cerebral ischemia; thus, strategies focused on maintaining CTSD function in neurons are potentially novel therapeutic approaches to prevent neuronal death in stroke. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; FTD: frontotemporal dementia, HD: Huntington disease; LAMP1: lysosomal associated membrane protein 1; LSD: lysosomal storage disease; MCAO: middle cerebral artery occlusion; OGD: oxygen glucose deprivation; OGR: oxygen glucose resupply; PD: Parkinson disease; SQSMT1: sequestosome 1; TCA: trichloroacetic acid; TTC: triphenyl tetrazolium chloride.

Background: The Cathepsins family, including cathepsin B and cathepsin D, potentially affects the entire processes involved in atherosclerosis. Although coronary heart disease (CHD) has been widely studied as the basis of Sudden Cardiac Death (SCD), the relationship between CHD and CTSB/D remains unclear., Methods: We screened for differentially expressed proteins (DEPs) associated with autophagy by limma package in R. For the genes corresponding to the DEPs after screening, we used various databases to carry out functional enrichment of related DEGs to explore their possible influence on a specific aspect of the disease. Functional enrichment analysis of DEGs was performed by DAVID, Metascape and GSEA. STRING and Cytoscape were obtained the hub genes, the analysis of interaction networks through the GENMANIA and Networkanalyst. Western Blot was used to validate the protein expression level of target genes. TF and miRNA prediction were performed using Networkanalyst and visualized using Cytoscape., Results: The expression levels of members of the cathepsin family were up regulated in CHD tissues compared with the control. GO and KEGG revealed that cathepsin was markedly enriched in endopeptidase activities, immune responses, lysosome pathways, et al. The correlation analysis showed that in patients with CHD, the CTSB/CTSD expression were negatively correlated with ATG4D and BNIP3, but positively with BCL2L1, CAPNS1, and TP53. In the TF-mRNA-miRNA network, has-miR-24-3p and has-miR-128-3p had higher degrees, CTSB/CTSD could be targeted by them., Conclusions: Our findings elucidated the expression and regulatory role of cathepsins in coronary heart disease induced SCD and might further explore the potential mechanisms of autophagy in CHD.

CTSD/CathD/CATD (cathepsin D) is a lysosomal aspartic protease. A distinguishing characteristic of CTSD is its dual functions of promoting cell proliferation via secreting a pro-enzyme outside the cells as a ligand, and promoting apoptosis via the mature form of this enzyme inside cells; however, the regulation of its secretion, expression, and maturation is undetermined. Using the lepidopteran insect Helicoverpa armigera , a serious agricultural pest, as a model, we revealed the dual functions and regulatory mechanisms of CTSD secretion, expression, and maturation. Glycosylation of asparagine 233 (N233) determined pro-CTSD secretion. The steroid hormone 20-hydroxyecdysone (20E) promoted CTSD expression. Macroautophagy/autophagy triggered CTSD maturation and localization inside midgut cells to activate CASP3 (caspase 3) and promote apoptosis. Pro-CTSD was expressed in the pupal epidermis and was secreted into the hemolymph to promote adult fat body endoreplication/endoreduplication, cell proliferation, and association. Our study revealed that the differential expression and autophagy-mediated maturation of CTSD in tissues determine its roles in apoptosis and cell proliferation, thereby determining the cell fates of tissues during lepidopteran metamorphosis. Abbreviations: 20E: 20-hydroxyecdysone; 3-MA: 3-methyladenine; ACTB/β-actin: actin beta; AKT: protein kinase B; ATG1: autophagy-related 1; ATG4: autophagy-related 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG14: autophagy-related 14; BSA: bovine serum albumin; CASP3: caspase 3; CQ: choroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DPBS: dulbecco's phosphate-buffered saline; DsRNA: double-stranded RNA; EcR: ecdysone receptor; EcRE: ecdysone response element; EdU: 5-ethynyl-2´-deoxyuridine; G-m-CTSD: glycosylated-mautre-CTSD; G-pro-CTSD: glycosylated-pro-CTSD; HaEpi: Helicoverpa armigera epidermal cell line; HE staining: hematoxylin and eosin staining; IgG: immunoglobin G; IM: imaginal midgut; JH: juvenile hormone; Kr-h1: krueppel homologous protein 1; LM: larval midgut; M6P: mannose-6-phosphate; PBS: phosphate-buffered saline; PCD: programmed cell death; PNGase: peptide-N-glycosidase F; RFP: red fluorescent protein; RNAi: RNA interference; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SYX17: syntaxin 17; USP1: ultraspiracle isoform 1.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods : The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd -/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro , cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca 2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The major lysosomal proteases, Cathepsin B (CTSB), Cathepsin D (CTSD) and Cathepsin L (CTSL), are implicated in autophagic activity. To investigate the role of each cathepsin in the exocrine pancreas, we generated mice in which the pancreas was specifically deficient in Ctsb, Ctsd and Ctsl. Each of these gene knockout (KO) and Ctsb;Ctsl and Ctsd;Ctsl double-knockout (DKO) mice were almost normal. However, we found cytoplasmic degeneration in the pancreatic acinar cells of Ctsb;Ctsd DKO mice, similar to autophagy related 5 (Atg5) KO mice. LC3 and p62 (autophagy markers) showed remarkable accumulation and the numbers of autophagosomes and autolysosomes were increased in the pancreatic acinar cells of Ctsb;Ctsd DKO mice. Moreover, these Ctsb;Ctsd DKO mice also developed chronic pancreatitis (CP). Thus, we conclude that both Ctsb and Ctsd deficiency caused impaired autophagy in the pancreatic acinar cells, and induced CP in mice.

Vision loss is among the characteristic symptoms of neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative lysosomal storage disorder. Here, we performed an in-depth analysis of retinal degeneration at the molecular and cellular levels in mice lacking the lysosomal aspartyl protease cathepsin D, an animal model of congenital CLN10 disease. We observed an early-onset accumulation of storage material as indicated by elevated levels of saposin D and subunit C of the mitochondrial ATP synthase. The accumulation of storage material was accompanied by reactive astrogliosis and microgliosis, elevated expression of the autophagy marker sequestosome 1/p62 and a dysregulated expression of several lysosomal proteins. The number of cone photoreceptor cells was reduced as early as at postnatal day 5. At the end stage of the disease, the outer nuclear layer was almost atrophied, and all cones were lost. A significant loss of rod and cone bipolar cells, amacrine cells and ganglion cells was found at advanced stages of the disease. Results demonstrate that cathepsin D deficiency results in an early-onset and rapidly progressing retinal dystrophy that involves all retinal cell types. Data of the present study will serve as a reference for studies aimed at developing treatments for retinal degeneration in CLN10 disease.

The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB -/- HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cathepsin D, an aspartyl protease, is an attractive therapeutic target for various diseases, primarily cancer and osteoarthritis. However, despite several small molecule cathepsin D inhibitors being developed, that are highly potent, most of them show poor microsomal stability, which in turn limits their clinical translation. Herein, we describe the design, optimization and evaluation of a series of novel non-peptidic acylguanidine based small molecule inhibitors of cathepsin D. Optimization of our hit compound 1a (IC 50  = 29 nM) led to the highly potent mono sulphonamide analogue 4b (IC 50  = 4 nM), however with poor microsomal stability (HLM: 177 and MLM: 177 μl/min/mg). To further improve the microsomal stability while retaining the potency, we carried out an extensive structure-activity relationship screen which led to the identification of our optimised lead 24e (IC 50  = 45 nM), with an improved microsomal stability (HLM: 59.1 and MLM: 86.8 μl/min/mg). Our efforts reveal that 24e could be a good starting point or potential candidate for further preclinical studies against diseases where Cathepsin D plays an important role., (Copyright © 2020 Elsevier Ltd. All rights reserved.)