Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aß accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

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.

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.

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.

Synthesis and biological evaluation of selective Pepstatin based trifluoromethylated inhibitors of Cathepsin D.

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.

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.

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.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Lysosomal membrane stabilization by imipramine attenuates gentamicin-induced renal injury: Enhanced LAMP2 expression, down-regulation of cytoplasmic cathepsin D and tBid/cytochrome c/cleaved caspase-3 apoptotic signaling.

Nephrotoxicity is a serious complication commonly encountered with gentamicin (GTM) treatment. Permeabilization of lysosomes with subsequent cytoplasmic release of GTM and cathepsins is considered a crucial issue in progression of GTM toxicity. This study was designed to evaluate the prospective defensive effect of lysosomal membrane stabilization by imipramine (IMP) against GTM nephrotoxicity in rats. GTM (30 mg/kg/h) was intraperitoneally administered over 4 h daily (120 mg/kg/day) for 7 days. IMP (30 mg/kg/day) was orally administered for 14 days; starting 7 days before and then concurrently with GTM. On 15th day, samples (urine, blood, kidney) were collected to estimate biomarkers of kidney function, lysosomal stability, apoptosis, and inflammation. IMP administration to GTM-treated rats ameliorated the disruption in lysosomal membrane stability induced by GTM. That was evidenced by enhanced renal protein expressions of LAMP2 and PI3K, but reduced cathepsin D cytoplasmic expression in kidney sections. Besides, IMP guarded against apoptosis in GTM-treated rats by down-regulation of the pro-apoptotic (tBid, Bax, cytochrome c) and the effector cleaved caspase-3 expressions, while the anti-apoptotic Bcl-2 expression was enhanced. Additionally, the inflammatory cascade p38 MAPK/NF-κB/TNF-α was attenuated in GTM + IMP group along with marked improvement in kidney function biomarkers, compared to GTM group. These findings were supported by the obvious improvement in histological architecture. Furthermore, in vitro enhancement of the antibacterial activity of GTM by IMP confers an additional benefit to their combination. Conclusively, lysosomal membrane stabilization by IMP with subsequent suppression of tBid/cytochrome c/cleaved caspase-3 apoptotic signaling could be a promising protective strategy against GTM nephrotoxicity.

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.

Cathepsin D Attenuates the Proliferation of Vascular Smooth Muscle Cells Induced by the AGE/RAGE Pathway by Suppressing the ERK Signal.

BACKGROUND: In this study, we aimed to clarify the role and mechanism by which Cathepsin D (CTSD) mediates the advanced glycation end products (AGEs)-induced proliferation of vascular smooth muscle cells (VSMCs). METHODS: We conducted a Western blotting assay and co-immunoprecipitation assay to detect the expression of target proteins and the interaction between different proteins. Cell Counting Kit-8 (CCK-8) assay and 5- ethynyl-2'-deoxyuridine (EdU) were used to evaluate the proliferation. RESULTS: AGEs significantly promoted phenotypic switching and proliferation of VSMCs in a concentration-dependent manner. This effect of AGEs was accompanied by inhibition of CTSD. Both the proliferation of VSMCs and inhibition of CTSD induced by AGEs could be attenuated by the specific inhibitor of the receptor for advanced glycation end products (RAGE), FPS-ZM1. Overexpression of CTSD significantly alleviated these effects of AGEs on VSMCs. The mechanism of CTSD action in VSMCs was also explored. Overexpression of CTSD reduced the activation of p-ERK caused by AGEs. By contrast, the knockdown of CTSD, elicited using a plasmid containing short hairpin RNA (shRNA) against CTSD, further increased the activation of p-ERK compared to AGEs alone. Additionally, co-immunoprecipitation studies revealed an endogenous interaction between CTSD, a protease, and p-ERK, its potential substrate. CONCLUSION: It has been demonstrated that CTSD downregulates the level of phosphorylated ERK by degrading its target, and this interaction plays a critical role in the proliferation of VSMCs induced by the AGE/RAGE axis. These results provide a novel insight into the prevention and treatment of vascular complications in diabetes.

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.

Fingerprinting of Proteases, Protease Inhibitors and Indigenous Peptides in Human Milk.

The presence of proteases and their resulting level of activity on human milk (HM) proteins may aid in the generation of indigenous peptides as part of a pre-digestion process, of which some have potential bioactivity for the infant. The present study investigated the relative abundance of indigenous peptides and their cleavage products in relation to the abundance of observed proteases and protease inhibitors. The proteomes and peptidomes in twelve HM samples, representing six donors at lactation months 1 and 3, were profiled. In the proteome, 39 proteases and 29 protease inhibitors were identified in 2/3 of the samples. Cathepsin D was found to be present in higher abundance in the proteome compared with plasmin, while peptides originating from plasmin cleavage were more abundant than peptides from cathepsin D cleavage. As both proteases are present as a system of pro- and active- forms, their activation indexes were calculated. Plasmin was more active in lactation month 3 than month 1, which correlated with the total relative abundance of the cleavage product ascribed to plasmin. By searching the identified indigenous peptides in the milk bioactive peptide database, 283 peptides were ascribed to 10 groups of bioactivities. Antimicrobial peptides were significantly more abundant in month 1 than month 3; this group comprised 103 peptides, originating from the ß-CN C-terminal region.

A novel function by cathepsin D in degradation of nucleic acids.

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. Ca2+ and Mg2+ 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 104:1. Kinetic parameters of Km and kcat/Km value were (42 ± 1) µM and (1.62 ± 0.1) × 10-2 s-1mM-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.

Increased Osteoclastogenesis in Absence of TG2 Is Reversed by Transglutaminase Inhibition-Evidence for the Role for TG1 in Osteoclast Formation.

Osteoclasts are multinucleated, bone-resorbing giant cells derived from monocyte-macrophage cell lines. Increased bone resorption results in loss of bone mass and osteoporosis. Osteoclast and bone marrow macrophages have been shown to express three TG enzymes (TG2, Factor XIII-A, and TG1) and TG activity to regulate osteoclast differentiation from bone marrow macrophages in vitro. In vivo and in vitro studies have demonstrated that the deletion of TG2 causes increased osteoclastogenesis and a significant loss of bone mass in mice (Tgm2-/- mice). Here, we confirm that TG2 deficiency results in increased osteoclastogenesis in vitro and show that this increase can be reversed by a TG inhibitor, NC9, suggesting that other TGs are responsible for driving osteoclastogenesis in the absence of TG2. An assessment of total TG activity with 5-(biotinamido)-pentylamine, as well as TG1 and FXIII-A activities using TG-specific Hitomi peptides (bK5 and bF11) in Tgm2-/- bone marrow flushes, bone marrow macrophages, and osteoclasts, showed a significant increase in total TG activity and TG1 activity. Factor XIII-A activity was unchanged. Aspartate proteases, such as cathepsins, are involved in the degradation of organic bone matrix and can be produced by osteoclasts. Moreover, Cathepsin D was shown in previous work to be increased in TG2-null cells and is known to activate TG1. We show that Pepstatin A, an aspartate protease inhibitor, blocks osteoclastogenesis in wild-type and Tgm2-/- cells and decreases TG1 activity in Tgm2-/- osteoclasts. Cathepsin D protein levels were unaltered in Tgm2-/-cells and its activity moderately but significantly increased. Tgm2-/- and Tgm2+/+ bone marrow macrophages and osteoclasts also expressed Cathepsin E, and Renin of the aspartate protease family, suggesting their potential involvement in this process. Our study brings further support to the observation that TGs are significant regulators of osteoclastogenesis and that the absence of TG2 can cause increased activity of other TGs, such as TG1.

Cathepsin D mediates prenatal caffeine exposure-caused NAFLD susceptibility in male rat offspring by regulating autophagy.

Epidemiological evidence has revealed that non-alcoholic fatty liver disease (NAFLD) harbors an intrauterine origin. Autophagy is known to be involved in the protective mechanism in the development of adult NAFLD, but whether it engages in the occurrence of fetal-originated NAFLD remains unclear. In this study, a rat model of fetal-originated NAFLD was established by giving a high-fat diet or chronic stress after birth on prenatal caffeine exposure (PCE) male offspring. The alterations of liver morphologic analysis, lipid metabolism, and autophagy before and after birth were determined to confirm autophagy mechanism, NAFLD susceptibility, and intrauterine origin in PCE male adult offspring. Our results revealed that PCE-induced intrauterine high concentration of corticosterone exposure blocked autophagic flux by inhibiting cathepsin D expression in hepatocytes, leading to ß-oxidation inhibition and lipid accumulation in the liver. Moreover, high concentration of corticosterone upregulated miR-665 by activating the glucocorticoid receptor to suppress cathepsin D, thus causing lysosomal degradation dysfunction and autophagy flux blockade. Notably, hepatic overexpression of cathepsin D could reverse PCE-induced postnatal NAFLD susceptibility in male rat offspring. This study elucidates the epigenetic programming mechanism of intrauterine autophagy-related fetal-originated NAFLD susceptibility, and identifies cathepsin D as its early intervention target, providing an experimental basis for exploring early prevention and treatment strategies for fetal-originated NAFLD.

Effects of Vitamin D3 Supplementation and Aerobic Training on Autophagy Signaling Proteins in a Rat Model Type 2 Diabetes Induced by High-Fat Diet and Streptozotocin.

The aim of this study was to investigate the combined effects of vitamin D3 supplementation and aerobic training on regulating the autophagy process in rats with type 2 diabetic induced by a high-fat diet and streptozotocin. A total of 40 Wistar rats were divided into five groups: normal control (NC), diabetic control (DC), diabetic + aerobic training (DAT), diabetic + vitamin D3 (DVD), and diabetic + aerobic training + vitamin D3 (DVDAT). The rats underwent eight weeks of aerobic training with an intensity of 60% maximum running speed for one hour, along with weekly subcutaneous injections of 10,000 units of vitamin D3. The protein levels of different autophagy markers were assessed in the left ventricular heart tissue. The results showed that the protein levels of AMPK, pAMPK, mTOR, and pmTOR were significantly lower in the DC group compared to the NC group. Conversely, the levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D proteins were significantly higher in the DC group. However, the interventions of aerobic training and vitamin D3 supplementation, either individually or in combination, led to increased levels of AMPK, pAMPK, mTOR, and pmTOR, and decreased levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D (p < 0.05). Additionally, the aerobic capacity in the DAT and DVDAT groups was significantly higher compared to the NC, DC, and DVD groups (p < 0.05). These findings suggest that type 2 diabetes is associated with excessive autophagy in the left ventricle. However, after eight weeks of vitamin D3 supplementation and aerobic training, a significant reduction in excessive autophagy was observed in rats with type 2 diabetes.

Ginkgo biloba extract (EGb-761) confers neuroprotection against ischemic stroke by augmenting autophagic/lysosomal signaling pathway.

Ginkgo biloba extract (EGb-761) is well-recognized to have neuroprotective properties. Meanwhile, autophagy machinery is extensively involved in the pathophysiological processes of ischemic stroke. The EGb-761 is widely used in the clinical treatment of stroke patients. However, its neuroprotective mechanisms against ischemic stroke are still not fully understood. The present study was conducted to uncover whether the pharmacological effects of EGb-761 can be executed by modulation of the autophagic/lysosomal signaling axis. A Sprague-Dawley rat model of ischemic stroke was established by middle cerebral artery occlusion (MCAO) for 90 min, followed by reperfusion. The EGb-761 was then administered to the MCAO rats once daily for a total of 7 days. Thereafter, the penumbral tissues were acquired to detect proteins involved in the autophagic/lysosomal pathway including Beclin1, LC-3, SQSTM1/p62, ubiquitin, cathepsin B, and cathepsin D by western blot and immunofluorescence, respectively. Subsequently, the therapeutic outcomes were evaluated by measuring the infarct volume, neurological deficits, and neuron survival. The results showed that the autophagic activities of Beclin1 and LC3-II in neurons were markedly promoted by 7 days of EGb-761 therapy. Meanwhile, the autophagic cargoes of insoluble p62 and ubiquitinated proteins were effectively degraded by EGb-761-augmented lysosomal activity of cathepsin B and cathepsin D. Moreover, the infarction size, neurological deficiencies, and neuron death were also substantially attenuated by EGb-761 therapy. Taken together, our study suggests that EGb-761 exerts a neuroprotective effect against ischemic stroke by promoting autophagic/lysosomal signaling in neurons at the penumbra. Thus, it might be a new therapeutic target for treating ischemic stroke.

Myricitrin from bayberry as a potential inhibitor of cathepsin-D: Prospects for squamous lung carcinoma prevention.

Cathepsin-D (CATD) inhibitors' design and development drawn interest due to their potential therapeutic applications in managing different cancer types, including lung cancer. This study investigated myricitrin, a flavonol-3-O-rhamnoside, for its binding affinity to CATD. Molecular docking experiments revealed a strong binding affinity (-7.8 kcal/mol). Molecular dynamics (MD) simulation confirmed the complex's stability, while enzyme activity studies showed inhibitory concentration (IC50) of 35.14 ± 6.08 µM (in cell-free) and 16.00 ± 3.48 µM (in cell-based) test systems. Expression analysis indicated downregulation of CATD with a fold change of 1.35. Myricitrin demonstrated antiproliferative effects on NCIH-520 cells [IC50: 64.11 µM in Sulphorhodamine B (SRB), 24.44 µM in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], but did not affect healthy CHANG cells. It also prolonged the G2/M phase (at 10 µM: 1.19-fold; at 100 µM: 1.13-fold) and increased sub-diploid population by 1.35-fold. Based on the analysis done using SwissADME program, it is predicted that myricitrin is not a cytochrome p450s (CYPs) inhibitor, followed the rule of Ghose and found not permeable to the blood-brain barrier (BBB) which suggests it as a safe molecule. In summary, the experimental findings may establish the foundation for myricitrin and its analogues to be used therapeutically in CATD-mediated lung cancer prevention.