Cathepsin D is essential for the degradomic shift of macrophages required to resolve liver fibrosis.

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 (CtsDΔMyel) 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 CtsDΔMyel livers. Besides, CtsDΔMyel 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.

Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.

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-synucleinSer129 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-synucleinSer129 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.

Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

Impaired cathepsin D in retinal pigment epithelium cells mediates Stargardt disease pathogenesis.

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.

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

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 ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-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.

Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.

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 promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation.

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.

Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques.

Alzheimer's disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aß) peptides. The extracellular deposition of Aß peptides in human AD brain causes neuronal death. Therefore, it has been found that Aß 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.

Enhancement of Acetate-Induced Apoptosis of Colorectal Cancer Cells by Cathepsin D Inhibition Depends on Oligomycin A-Sensitive Respiration.

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.

Cycloheptylprodigiosin from marine bacterium Spartinivicinus ruber MCCC 1K03745T induces a novel form of cell death characterized by Golgi disruption and enhanced secretion of cathepsin D in non-small cell lung cancer cell lines.

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 1K03745T, and its anticancer property was investigated. It exhibits remarkable cytotoxicity against a panel of human lung cancer cell lines, with the IC50 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.

Specific photodamage on HT-29 cancer cells leads to endolysosomal failure and autophagy blockage by cathepsin depletion.

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Expression of Cathepsin D in early-stage breast cancer and its prognostic and predictive value.

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.

Monocytes Release Pro-Cathepsin D to Drive Blood-to-Brain Transcytosis in Diabetes.

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.

Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified. Here we show that extracellular α-Syn caused S1P receptor type 3 (S1PR3) uncoupled from G protein in HeLa cells. Further studies indicated that α-Syn treatment reduced cathepsin D activity while enhancing the secretion of immature pro-cathepsin D into cell culture medium, suggesting that lysosomal delivery of cathepsin D was disturbed. Actually, extracellular α-Syn attenuated the retrograde trafficking of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor, which is under the regulation of S1PR3. These findings shed light on the understanding of dissemination of the PD pathology, that is, the mechanism underlying how extracellular α-Syn secreted from pathogenic cells causes lysosomal dysfunction of the neighboring healthy cells, leading to propagation of the disease.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

Cathepsin D promotes polarization of tumor-associated macrophages and metastasis through TGFBI-CCL20 signaling.

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.

Lnc-HIBADH-4 Regulates Autophagy-Lysosome Pathway in Amyotrophic Lateral Sclerosis by Targeting Cathepsin D.

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.

Lysosomes, caspase-mediated apoptosis, and cytoplasmic activation of P21, but not cell senescence, participate in a redundant fashion in embryonic morphogenetic cell death.

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-ß-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 ß-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-ß-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-ß-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling.

Enhanced Osteocyte Differentiation: Cathepsin D and L Secretion by Human Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases.

Expression and significance of cathepsin C and cathepsin D during pregnancy and Preeclampsia.

BACKGROUND: Cathepsin C (Cat C) is involved in the inflammatory-immune system and can be degraded by cathepsin D (Cat D). Preeclampsia (PE) and the inflammation-immunity relationship is currently a hot research topic, but there are still few studies. The aim was to investigate the expression and significance of Cat C and D in the serum of nonpregnant women, patients in various stages of pregnancy and patients with PE, and in the placenta of patients with normal pregnancy and PE. METHODS: Sixty young healthy nonpregnant women were selected: 180 normal pregnant women, including 60 each in the first, second, and third trimesters, and 100 women with PE, including 39 women with severe preeclampsia. The levels of Cat C and D in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the expression levels of Cat C and D in placentas were detected by immunohistochemistry (IHC). RESULTS: The serum of Cat C in the first trimester was significantly lower than that in the nonpregnant group (P < 0.001), whereas Cat D was significantly higher than that in the nonpregnant group (P < 0.01). The levels of Cat C and D in the second trimester and third trimester were significantly higher than those in the first trimester (P < 0.05), but there was no significant difference in Cat C and D between the second trimester and third trimester. The levels of Cat C in the serum and placentas of patients with PE were significantly higher than those in the third trimester (P < 0.001) and positively correlated with the severity of PE (P < 0.001), whereas the levels of Cat D in the serum and placentas of patients with PE were significantly lower than those in the third trimester (P < 0.001) and negatively correlated with the severity of PE (P < 0.001). Age, primigravida proportion, and body mass index were significantly higher in the PE group than in the control group (P < 0.05), which were high-risk factors for PE. CONCLUSIONS: Cat C and D are associated with the maintenance of normal pregnancy. In patients with preeclampsia, a significant increase in Cat C and a significant decrease in Cat D levels may lead to the occurrence and development of preeclampsia.