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The objective of this study was to investigate the differential role of cathepsin B, L, D and calpain in degradation and disassembly of myofilament. Myofibrillar protein of grass carp (Ctenopharyngodon idella) was incubated with proteases monotonously, simultaneously or sequentially. Subsequently, protein degradation were detected using SDS-PAGE and myofilament disassembly induced by changes of non-covalent interactions were measured through SDS-PAGE using l-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) as a zero length cross-linker. Additionally, content of heat shock proteins which functioned in stabilizing assembly architecture of myofibrillar protein was determined. Results showed that calpain and cathepsin B, calpain and cathepisn L could act in a stepwise and complimentary manner to synergistically dissociate and degrade myofibrillar protein. In synergistic action, cathepsin B disrupted the thick filament assembly through lowering the UNC45 and HSP90 concentration in myofibrillar protein, facilitating the degradation of dissociated MHC by calpain. Meanwhile, Cathepsin L was shown to preferentially remove the actin from thin filament via lowering the content of HSP27 and αb-crystallin, to create dissociated actin as substrate supply for calpain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The hyaloid vasculature constitutes a transitory system nourishing the internal structures of the developing eye, but the mechanism of vascular regression and its cell biological characteristics are not fully understood. The present study aimed to reveal the specificity of the hyaloid vessels by a systematic immunohistochemical approach for marker substances of myeloid cells and the extracellular matrix (ECM) in neonatal mice. Macrophages immunoreactive for F4/80, cathepsin D, and LYVE-1 gathered around the vasa hyaloidea propria (VHP), while small round cells in vascular lumen of VHP were selectively immunoreactive for galectin-3; their segmented nuclei and immunoreactivities for Ly-6G, CD11b, and myeloperoxidase indicated their neutrophilic origin. VHP possessed thick ECM and a dense pericyte envelope as demonstrated by immunostaining for laminin, type IV collagen, integrin β1, and NG2. The galectin-3 + cells loosely aggregated with numerous erythrocytes in the lumen of hyaloid vessels in a manner reminiscent of vascular congestion. Galectin-3 is known to polymerize and form a complex with ECM and NG2 as well as recruit leukocytes on the endothelium. Observation of galectin-3 KO mice implicated the involvement of galectin-3 in the regression of hyaloid vasculature. Since macrophages may play central roles including blocking of the blood flow and the induction of apoptosis in the regression, galectin-3 + neutrophils may play a supportive role in the macrophage-mediated involution of the hyaloid vascular system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Activation of lysosomal proteolysis and increased coenzyme Q10 level were revealed in the liver and brain of rats with individual typological features under conditions of food deprivation. Changes in cathepsin B activity in these tissues were different in behaviorally active and passive animals. We observed indirect modulating effect of coenzyme Q10 in the regulation of activity of lysosomal endopeptidases in the brain of animals with different behavior during adaptation to metabolic stress.

Anti-Vascular Endothelial Growth Factor (VEGF) therapy is given repeatedly for an extended period of time to patients when treated for age-related macular degeneration. While short-term effects of anti-VEGF agents on retinal pigment epithelial (RPE) cells have been investigated, the effects of long-term and repeated treatment on these cells are scarce. In this study, we have investigated the effects of anti-VEGF treatment after long-term, repeated treatment on cell aging and morphology. The experiments were conducted in primary porcine RPE cells passage one and two. Cells were treated with 125 μg/ml bevacizumab, ranibizumab, aflibercept or rituximab once a week for 1 day, 4 days, 7 days, 4 weeks and 12 weeks. Cell survival was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) and trypan blue exclusion assay. Activity of β-galactosidase was assessed in a commercially available assay. Influence of these compounds was investigated on the expression of cathepsin D and amyloid β, and the expression and phosphorylation of mechanistic target of rapamycin (mTOR), all proteins involved in senescence and aging, in Western blot. The secretion of Pigment Epithelium Derived Factor (PEDF) and Transforming Growth Factor (TGF)-β was investigated in Enzyme-linked Immunosorbent Assay (ELISA). The cellular morphology was investigated with electron microscopy, investigating the number and area of mitochondria and autophagosomes. Statistical analysis was conducted using a mixed linear model. Weekly treatment up to 12 weeks displayed no toxic effects on RPE cells in any of the substances tested. Ranibizumab showed a significant increase in β-galactosidase signal on day 4 (p < 0.05) and 7 (p < 0.05) after treatment. In long-term, however, ranibizumab displayed no significant difference to untreated cells. Bevacizumab displayed a significant reduction of the β-galactosidase signal after 12 weeks (p < 0.05). Aflibercept significantly decreased β-galactosidase after 1 day (p < 0.01) and 12 weeks (p < 0.05). Rituximab and bevacizumab also decreased β-galactosidase signal after 12 weeks (p < 0.05). The expression of mTOR, phospho-mTOR, amyloid β and cathepsin D was not significantly altered by any of the compounds tested. RPE cells secreted considerate amounts of TGF-β. Bevacizumab treated cells showed significantly lower TGF-β secretion than ranibizumab and rituximab (p < 0.05). In contrast, only small amounts of PEDF were secreted which were not altered by any substance tested. Ultrastructural analysis showed no alterations in mitochondria after long-term treatment with either substance. Autophagosomes were not reduced by long-term anti-VEGF treatment compared to control. However, the area of autophagosomes in bevacizumab and aflibercept treated cells was significantly less compared to both ranibizumab and rituximab treated cells (all p < 0.05). Taken together, weekly treatment with VEGF-antagonists up to 3 months does not induce premature aging in primary RPE cells in any tested compound. A significant difference can be found between bevacizumab and aflibercept on the one hand, and ranibizumab (and rituximab) on the other hand, with more autophagosomal area in ranibizumab and (rituximab). Taken together, our data provide indications for long-term safety of anti-VEGF compounds. Further research is warranted., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The deposition of advanced glycation end products (AGEs) is accelerated in photoaged skin, but the underlying mechanisms remain elusive. Intracellular degradation has been recently considered to play an important role in AGEs removal. Although lysosomal cathepsin D (CatD), B (CatB), L(CatL) and proteasomes are found to degrade internalized AGEs, it remains unknown which protease degrades internalized AGEs in human dermal fibroblasts (HDFs), and whether a decrease in intracellular degradation contributes to enhanced AGEs deposition in photoaged skin., Objective: This study aims to investigate the specific proteases that contribute to intracellular AGEs degradation in HDFs and regulate AGEs accumulation in photoaged skin., Methods: Repetitive UVA irradiation was used to induce primary HDF photoaging in vitro. Uptake and degradation of AGE-BSA were verified and compared between photoaged and non-photoaged fibroblasts with flow cytometry, ELISA and confocal microscopy. Proteasomal and lysosomal activity, expression of CatD, CatB and CatL were also investigated between photoaged and non-photoaged fibroblasts. Further, the effect of protease inhibitors and CatD overexpression via lentiviral transduction on AGE-BSA degradation was analyzed. Finally, the correlation between CatD expression and AGEs accumulation in sun-exposed and sun-protected skin of people from different age was studied with immunohistochemistry., Results: Fibroblasts underwent photoaging in vitro after repetitive UVA irradiation. AGE-BSA was taken up by both photoaged and non-photoaged fibroblasts, but its degradation was significantly decreased in photoaged cells than that of non-photoaged cells. Although the activity of proteasome, CatB, Cat L and Cat D was significantly reduced in photoaged fibroblasts compared to that of non-photoaged cells, and the expression of CatB, CatL and CatD was profoundly attenuated in photoaged fibroblasts, inhibiting proteasome, CatB and CatL did not affect AGE-BSA degradation in HDFs. In contrast, inhibiting CatD activity dose-dependently decreased AGE-BSA degradation; whereas CatD overexpression significantly increased AGE-BSA degradation. Importantly, AGEs accumulation in photo-damaged skin in vivo was inversely correlated with CatD expression., Conclusion: CatD plays a major role in intracellular AGEs degradation. Decreased CatD expression and activity impairs intracellular AGEs degradation in photoaged fibroblasts, which may contribute to accelerated AGEs deposition in photoaged skin. The present study provides a potentially novel molecular basis for antiphotoaging therapy., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Cathepsins have been described in the pathogenesis of abdominal aortic aneurysm (AAA), their exact role, especially in collagen degradation, is still unclear. The aim of the present study was therefore to analyse relevant cathepsins in human AAA tissue samples in relation to collagen I, III, and their degradation products., Materials and Methods: Samples from 37 AAA patients obtained from elective open surgical repair and eight healthy non-aneurysmatic aortas from kidney donors were included. Expression of cathepsins B, D, K, L, S, cystatin C, collagen I and III, their degraded products C-Telopeptide of type 1 and 3 collagen (CTX-I, CTX-III), cellular markers for leukocytes (CD45), T cells (CD3), macrophage scavenger receptor-1 (MSR-1), synthetic, and contractile smooth muscle cells (SMCs) (smoothelin: SMTH, collagen I and III, myosin heavy chain: MHC, embryonic smooth muscle myosin heavy chain: SMemb) were determined at messenger RNA (mRNA) level, using SYBRGreen-based quantitative PCR and at protein level using enzyme-linked immunosorbent assay (ELISA)., Results: Expression of cathepsins B, D, L, and S at mRNA level was significantly elevated in AAA compared to control aorta (1.7-fold, p = 0.025; 2.5-fold, p = 0.002; 2.6-fold, p = 0.034; and 7.0-fold, p = 0.003). Expression of cathepsin S correlated significantly with leukocytes and macrophages (ρ = 0.398, p = 0.033 and ρ = 0.422, p = 0.020), synthetic SMCs were significantly associated with cathepsins B, D, and L (ρ = 0.522, p = 0.003; ρ = 0.431, p = 0.015 and ρ = 0.467, p = 0.008). At protein level, cathepsins B and S were elevated in AAA compared to controls (5.4-fold, p = 0.001 and 7.3-fold, p < 0.001). Significant correlations were observed between collagen I, its degraded product, and cathepsin S (r = -0.350, p = 0.034 and r = +0.504, p < 0.001). Expression of cathepsin B was associated with SMCs, expression of cathepsin S with inflammatory cells., Conclusions: Particularly cathepsin S was associated with the degradation product of collagen I and thus might be involved in the progression of AAA. Furthermore, cathepsin S correlated with inflammatory cells.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-β, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca 2+ /calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Exposure of Culex quinquefasciatus larvae to hypothermia or hyperthermia in relation to ecological temperature has resulted in alteration of enzyme activities related to maintenance of free amino acid pool and protein degradation. Sudden changes in water temperature have led to elevation of protein content particularly in molecular weight between 66 and 97.4 kDa. MALDI TOF analysis revealed the presence of putative uncharacterized protein and Phospholipase A2 activating protein in larvae subjected to 40°C for 1 hour which was not found in control and other temperature treated larvae. These proteins may be playing a role in survival of larvae at higher temperatures. Thermal shock has resulted in channeling of free amino acids for protein synthesis and elevation of amino acid catabolism through increased deamination and altered transamination, which resulted in a sharp decrease in free amino acid pool. Inhibition of Leucine amino peptidase and elevation of Cathepsin D, under the influence of thermal shock may be an adaptive response of larvae to prevent unnecessary degradation of protein and at the same time facilitated rapid internal re-organization which may accelerate pupation. Elevated activity of Phenol oxidase and its co-stimulator Trypsin -like Serine protease, along with increased expression of Phospholipase A2 activating protein is a well co-ordinated defense mechanism leading to increased immune response in stressed state. This study assumes significance because variation in environmental conditions such as global warming and their impact on mosquito physiology can influence efficiency of disease vector., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

For many neurodegenerative disorders, expression of a pathological protein by one cell type impedes function of other cell types, which in turn contributes to the death of the first cell type. In transgenic mice modelling Stargardt-like (STGD3) maculopathy, human mutant ELOVL4 expression by photoreceptors is associated with defects in the underlying retinal pigment epithelium (RPE). To examine how photoreceptors exert cytotoxic effects on RPE cells, transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates were studied one month prior (preclinical stage) to onset of photoreceptor loss (two months). TG photoreceptor outer segments presented to human RPE cells are recognized and internalized into phagosomes, but their digestion is delayed. Live RPE cell imaging pinpoints decreased numbers of acidified phagolysomes. In vivo, master regulator of lysosomal genes, transcription factor EB (TFEB), and key lysosomal enzyme Cathepsin D are both unaffected. Oxidative stress, as ruled out with high-resolution respirometry, does not play a role at such an early stage. Upregulation of CRYBA1/A3 and phagocytic cells (microglia/macrophages) interposed between RPE and photoreceptors support adaptive responses to processing delays. Impaired phagolysosomal maturation is observed in RPE of mice expressing human mutant ELOVL4 in their photoreceptors prior to photoreceptor death and associated vision loss.

Important differences have begun to emerge concerning the molecular profile of female and male breast cancer which may prove to be of therapeutic value. This review examined all the available data on the genomics of MBC. Most male cancers are ER+ve but without a corresponding increase in PR positivity and only a weaker association with estrogen-controlled markers such as PS2, HSP27 and Cathepsin-D. HER2 +ve cancers are rare in males and the role of androgen receptor is controversial. Although the Luminal A phenotype was the most frequent in both MBC and FBC, no Luminal B or HER2 phenotypes were found in males and the basal phenotype was very rare. Using hierarchical clustering in FBC, ERα clustered with PR, whereas in MBC, ERα associated with ERβ and AR. Based on limited data it appears that Oncotype DX is effective in determining recurrence risk in selected MBC. In future, tailored therapies based on genomics will probably yield the most promising approach for both MBC and FBC., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to investigate the role of cathepsin D (CathD) in central nervous system (CNS) myelination and its possible mechanism. By using CathD knockout mice in conjunction with immunohistochemistry, immunocytochemistry and western blot assays, the myelination of the CNS and the development of oligodendrocyte lineage cells in vivo and in vitro were observed. Endocytosis assays, real-time-lapse experiments and total internal reflection fluorescence microscopy were used to demonstrate the location and movement of proteolipid protein in oligodendrocyte lineage cells. In addition, the relevant molecular mechanism was explored by immunoprecipitation. The increase in Fluoromyelin Green staining and proteolipid protein expression was not significant in the corpus callosum of CathD -/- mice at the age of P11, P14 and P24. Proteolipid protein expression was weak at each time point and was mostly accumulated around the nucleus. The number of oligodendrocyte lineage cells (olig2+) and mature oligodendrocytes (CC1+) significantly decreased between P14 and P24. In the oligodendrocyte precursor cell culture of CathD -/- mice, the morphology of myelin basic protein-positive mature oligodendrocytes was simple while oligodendrocyte precursor cells showed delayed differentiation into mature oligodendrocytes. Moreover, more proteolipid protein gathered in late endosomes/lysosomes (LEs/Ls) and fewer reached the plasma membrane. Immunohistochemistry and immunoelectron microscopy analysis showed that CathD, proteolipid protein and VAMP7 could bind with each other, whereas VAMP7 and proteolipid protein colocalized with CathD in late endosome/lysosome. The findings of this paper suggest that CathD may have an important role in the myelination of CNS, presumably by altering the trafficking of proteolipid protein.

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: In the last decade, there has been an increase in the use of anti-angiogenic drugs as treatment for metastatic malignancies. However, use of these targeted therapies could induce both glomerular and tubular damage. Also during targeted therapy, the lysosomal protease cathepsin D is released from the tumour, which is inhibited by the protease inhibitor cystatin C. The aim of this study is to determine if use of cystatin C-estimated glomerular filtration rate (eGFR) is applicable to a patient cohort treated with targeted agents., Methods: A cohort of 80 patients with various malignancies were continuously recruited and prospectively analysed. Serum and urinary biochemical analytes for renal toxicities were assessed at different time points during treatment. The association between serum cystatin C and cathepsin D was also determined., Results: A decrease in serum cystatin C concentrations (1.03 versus 0.90 mg/L; P < 0.001), together with an increase in cystatin C-eGFR (71 versus 89 mL/min/1.73 m2; P = 0.002) was observed during therapy, compared with baseline. This decrease in cystatin C concentrations was correlated with cathepsin D (r = 0.307; P < 0.001), which was released from the tumour during targeted therapy. Further analysis demonstrated cathepsin D-mediated proteolysis of cystatin C in serum., Conclusions: Cystatin C concentrations were decreased during targeted therapy due to cathepsin D-mediated proteolysis. Cystatin C-eGFR is therefore not considered a suitable marker for assessing kidney function in oncology patients, and other techniques to estimate the GFR have to be applied in this patient population., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are pathogenic for familial Parkinson's disease. However, it is unknown whether levels of LRRK2 protein in the brain are altered in patients with LRRK2-associated Parkinson's disease. Because LRRK2 mutations are relatively rare, accounting for approximately 1% of all Parkinson's disease, we accessioned cases from five international brain banks to investigate levels of the LRRK2 protein, and other genetically associated Parkinson's disease proteins. Brain tissue was obtained from 17 LRRK2 mutation carriers (12 with the G2019S mutation and five with the I2020T mutation) and assayed by immunoblot. Compared to matched controls and idiopathic Parkinson's disease cases, we found levels of LRRK2 protein were reduced in the LRRK2 mutation cases. We also measured a decrease in two other proteins genetically implicated in Parkinson's disease, the core retromer component, vacuolar protein sorting associated protein 35 (VPS35), and the lysosomal hydrolase, glucocerebrosidase (GBA). Moreover, the classical retromer cargo protein, cation-independent mannose-6-phosphate receptor (MPR300, encoded by IGF2R), was also reduced in the LRRK2 mutation cohort and protein levels of the receptor were correlated to levels of LRRK2. These results provide new data on LRRK2 protein expression in brain tissue from LRRK2 mutation carriers and support a relationship between LRRK2 and retromer dysfunction in LRRK2-associated Parkinson's disease brain., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Suitable peptidases for biotechnological applications are those active at low temperature, in organic solvents, detergents or proteolytic additives. American lobster cathepsin D1 (CD1) is an enzyme highly efficient at 5-50°C and at pH 2.5-5.5. We assessed the effect of common industrial additives on CD1 activity. CD1 was isolated from lobster gastric fluid by chromatography. The proteolytic activity was measured using a fluorogenic specific substrate and the conformation by intrinsic fluorescence. Non-ionic detergents Tween-20 and Triton X-100 stabilize the peptidase activity. Ethanol, methanol and isopropanol [5-15% (v/v)] increased the enzyme activity up to 80%. The enzyme is active until 2.5M urea and is resistant to proteolysis by papain and renin. In this work, a crustacean peptidase that remains active when exposed to different chemical and proteolytic additives is reported, evincing that crustaceans are a good model for discovery of novel stable peptidases for future pharmaceutical, cosmetic and alimentary applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Acute pancreatitis is a complex disorder involving both premature intracellular protease activation and inflammatory cell invasion. An initiating event is the intracellular activation of trypsinogen by cathepsin B (CTSB), which can be induced directly via G protein-coupled receptors on acinar cells or through inflammatory cells. Here, we studied CTSB regulation by another lysosomal hydrolase, cathepsin D (CTSD), using mice with a complete (CTSD -/- ) or pancreas-specific conditional CTSD knockout (KO) (CTSD f/f /p48 Cre/+ ). We induced acute pancreatitis by repeated caerulein injections and isolated acinar and bone marrow cells for ex vivo studies. Supramaximal caerulein stimulation induced subcellular redistribution of CTSD from the lysosomal to the zymogen-containing subcellular compartment of acinar cells and activation of CTSD, CTSB, and trypsinogen. Of note, the CTSD KO greatly reduced CTSB and trypsinogen activation in acinar cells, and CTSD directly activated CTSB but not trypsinogen in vitro During pancreatitis in pancreas-specific CTSD f/f /p48 Cre/+ animals, markers of severity were reduced only at 1 h, whereas in the complete KO, this effect also included the late disease phase (8 h), indicating an important effect of extra-acinar CTSD on course of the disease. CTSD -/- leukocytes exhibited reduced cytokine release after lipopolysaccharide (LPS) stimulation, and CTSD KO also reduced caspase-3 activation and apoptosis in acinar cells stimulated with the intestinal hormone cholecystokinin. In summary, CTSD is expressed in pancreatic acinar and inflammatory cells, undergoes subcellular redistribution and activation during experimental pancreatitis, and regulates disease severity by potently activating CTSB. Its impact is only minimal and transient in the early, acinar cell-dependent phase of pancreatitis and much greater in the later, inflammatory cell-dependent phase of the disease., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background/aims: Autophagy is a well-known pathway to "clean" the misfolded mutant huntingtin protein (mHtt), which plays a considerable role in polyglutamine diseases. To date, there have been few studies of the choice of anesthetic during surgery in patients with polyglutamine diseases and evaluation of the effects and underlying mechanisms of anesthetics in these patients., Methods: GFP-Htt (Q74)-PC12 cells, which stably express green fluorescent protein-tagged Htt protein containing 74 glutamine repeating units, were used throughout this study. Cells were treated with 15 μM midazolam and 100 mM trehalose (positive control), and the induction of autophagy and autophagic degradation were assessed by detecting changes in autophagy-related proteins and substrates, and cell viability was assessed using the MTT assay. Overexpression of cathepsin D by plasmid transfection was used to restore midazolam-impaired autophagic degradation., Results: Midazolam increased intracellular mHtt levels in a time- and dose-dependent manner. Additionally, enhancing or blocking autophagic flux by trehalose or chloroquine could decrease or increase midazolam-induced mHtt elevation, respectively. Midazolam induced autophagy in the mTOR-dependent signaling pathway, but autophagic degradation was impaired, with a continuous rise in p62 and LC3 II levels and decrease in cathepsin D. However, overexpression of cathepsin D reversed the effects of midazolam. Midazolam led to a 20% decrease in GFP-Htt (Q74)-PC12 cell viability, which could be abrogated by overexpression of cathepsin D., Conclusions: Midazolam increased mHtt levels and decreased Htt (Q74)-PC12 cell viability via impairment of autophagic degradation, which could be restored by overexpression of cathepsin D., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Aim: Cathepsin D, one of the attractive targets in the treatment of breast cancer, has been implicated in HIV neuropathogenesis with potential proteolytic effects on chemokines. Methodology/result: Diverse modeling tools were used to reveal the key structural features affecting the inhibitory activities of 78 pepstatin A analogs. Analyses were performed to investigate the stability, rationality and fluctuation of the analogs. Results showed a clear correlation between the experimental and predicted activities of the analogs as well as the variation in their activities relative to structural modifications., Conclusion: The insight gained from this study offers theoretical references for understanding the mechanism of action of cathepsin D and will aid in the design of more potent and clinically-relevant drugs. Graphical abstract [Formula: see text].

Background: Early detection of Alzheimer's disease (AD) pathology is a serious challenge for both diagnosis and clinical trials. The aspartyl protease, Cathepsin D (CatD), is overexpressed in AD and could be a biomarker of disease. We have previously designed a unique contrast agent (CA) for dual-optical and magnetic resonance imaging of the activity of the CatD class of enzymes., Objective: To compare the uptake and retention of a novel, more sensitive, and clinically-translatable 68Ga PET tracer targeting CatD activity in 5XFAD mice and non-Tg littermates., Methods: The targeted CA consisted of an HIV-1 Tat cell penetrating peptide (CPP) conjugated to a specialized cleavage sequence targeting aspartyl cathepsins and a DOTA conjugate chelating 68Ga. PET images were acquired using a Siemens Inveon preclinical microPET in female Tg AD mice and non-Tg age matched female littermates (n = 5-8) following intravenous CA administration at 2, 6, and 9 months of age. Additionally, 18F fluorodeoxyglucose (FDG) PET imaging was performed at 10 months to measure glucose uptake., Results: The Tg mice showed significantly higher relative uptake rate of the targeting CA in the forebrain relative to hindbrain at all ages compared to controls, consistent with histology. In contrast, no differences were seen in CA uptake in other organs. Additionally, the Tg mice did not show any differences in relative uptake of FDG at 10 months of age in the forebrain relative to the hindbrain compared to age matched non-Tg controls., Conclusions: Elevated aspartryl cathepsin activity was detected in vivo in the 5XFAD mouse model of AD using a novel targeted PET contrast agent.

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.

Frontotemporal dementia (FTD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL). However, how dose-dependent PGRN mutations contribute to these two different diseases is not well understood. Using iPSC-derived human cortical neurons from FTD patients harboring PGRN mutations, we demonstrate that PGRN mutant neurons exhibit decreased nuclear TDP-43 and increased insoluble TDP-43, as well as enlarged electron-dense vesicles, lipofuscin accumulation, fingerprint-like profiles and granular osmiophilic deposits, suggesting that both FTD and NCL-like pathology are present in PGRN patient neurons as compared to isogenic controls. PGRN mutant neurons also show impaired lysosomal proteolysis and decreased activity of the lysosomal enzyme cathepsin D. Furthermore, we find that PGRN interacts with cathepsin D, and that PGRN increases the activity of cathepsin D but not cathepsins B or L. Finally, we show that granulin E, a cleavage product of PGRN, is sufficient to increase cathepsin D activity. This functional relationship between PGRN and cathepsin D provides a possible explanation for overlapping NCL-like pathology observed in patients with mutations in PGRN or CTSD, the gene encoding cathepsin D. Together, our work identifies PGRN as an activator of lysosomal cathepsin D activity, and suggests that decreased cathepsin D activity due to loss of PGRN contributes to both FTD and NCL pathology in a dose-dependent manner., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Objective: This study investigated potential associations between novel biomarkers for cardiovascular disease and other surrogate markers for health., Methods: Community sample of 170 (92 boys and 78 girls) children aged 8-11 years. Total fat mass (TBF) and abdominal fat (AFM) were measured by Dual-energy x-ray absorptiometry (DXA). Total body fat was also expressed as percentage of total body mass (BF%), and body fat distribution was calculated as AFM/TBF. Maximal oxygen uptake (VO2PEAK), systolic and diastolic blood pressure (SBP and DBP) and pulse pressure (PP) were measured. Echocardiography was performed. Left atrial size (LA) and left ventricular mass (LVM) were measured. A follow-up DXA scan was available in 152 children (84 boys and 68 girls). Frozen serum samples were analyzed for cystatin B, cathepsin L and cathepsin D., Results: Partial correlations between cystatin B versus lnTBF, lnBF%, lnAFM, AFM/TBF, VO2PEAK and PP were; r = 0.38, 0.36, 0.38, 0.29, -0.25 and 0.25, P = 0.001 or less for all. Weaker predominantly non-significant correlations were found for cathepsin L, whereas cathepsin D was not related to any surrogate markers for health. No significant correlations were found between biomarkers and change in body fat over 2 years., Conclusion: Findings from this community-based cohort of young children show that surrogate markers for cardiovascular disease such as total fat mass, percent body fat, abdominal fat, body fat distribution, maximal oxygen uptake and pulse pressure were all associated with cystatin B. This was not found for cathepsin L or cathepsin D.

Among Neuronal Ceroid Lipofuscinoses (NCLs), which are childhood fatal neurodegenerative disorders, the juvenile onset form (JNCL) is the most common. JNCL is caused by recessive mutations in the CLN3 gene. CLN3 encodes a lysosomal/endosomal transmembrane protein but its precise function is not completely known. We have previously reported that in baby hamster kidney (BHK) cells stably expressing myc-tagged human CLN3 (myc-CLN3), hyperosmotic conditions drastically increased myc-CLN3 mRNA and protein expression. In the present study, we analyzed the consequences of hyperosmolarity, and increased CLN3 expression on cathepsin D (CTSD) activity and prosaposin processing using BHK cells transiently or stably expressing myc-CLN3. We found that hyperosmolarity increased lysotracker staining of lysosomes, and elevated the levels of myc-CLN3 and lysosome-associated membrane protein-1 (LAMP1). Hyperosmolarity, independently of the expression level of myc-CLN3, decreased the levels of PSAP and saposin D, which are protein cofactors in sphingolipid metabolism. The lysosomal enzyme cathepsin D (CTSD) mediates the proteolytic cleavage of PSAP precursor into saposins A-D. Myc-CLN3 colocalized with CTSD and activity of CTSD decreased as myc-CLN3 expression increased, and clearly decreased under hyperosmotic conditions. Nevertheless, levels of CTSD measured by Western blotting were not altered under any studied condition. Our results suggest a direct involvement of CLN3 in the regulation of CTSD activity. J. Cell. Biochem. 118: 3883-3890, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Atherosclerosis is a common pathological basis of cardiovascular disease and remains the leading cause of mortality. Endothelial cell (EC) injury and autophagy dysfunction have been proved to contribute to the development of atherosclerosis. Recently, accumulating evidence confirms that microRNAs (miRNAs) have emerged as vital regulators and fine-tuners of various pathophysiological cellular impacts and molecular signaling pathways involved in atherosclerosis. Herein, the objective of the present study was to explore the biological function of miR-21 in oxidized low-density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs) injury and the underlying molecular mechanism. The results showed that ox-LDL treatment significantly decreased HAECs viability, increased caspase-3 activity, apoptosis ratio and Bax protein expression, and reduced Bcl-2 protein expression resulting in EC injuries. Simultaneously, ox-LDL treatment obviously reduced miR-21 level in a time-and dose-dependent manner. Notably, ox-LDL-induced EC injuries were abolished by miR-21 mimics transfection. In addition, miR-21 mimics alleviated ox-LDL-induced impaired autophagic flux as illustrated by the increases in LC3-II/LC3-I ratio and Beclin-1 protein expression, and the decrease in p62 protein expression in HAECs. Moreover, ox-LDL suppressed the expressions of lysosomal membrane protein (LAMP1) and cathepsin D proteins, and attenuated cathepsin D activity in HAECs, leading to lysosomal dysfunction, while these effects were also blocked by miR-21 mimics. These findings indicated that miR-21 restored impaired autophagic flux and lysosomal dysfunction, thereby attenuating ox-LDL-induced HAECs injuries., (Copyright © 2017. Published by Elsevier Inc.)

Maternal smoking during pregnancy is known to be related to adverse pregnancy results associated with trophoblast proliferation and cell cycle progression. Moreover, many previous studies have shown that cigarette smoke is correlated with human chorionic gonadotropin beta (hCG-β) subunit produced from syncytiotrophoblasts during pregnancy. Thus, we further investigated whether cigarette smoke extract (CSE) affects the cell proliferation, migration and endocrine hormone activity of JEG-3 human placental cancer cells. JEG-3 cell proliferation was significantly reduced by all CSEs in a concentration-dependent manner. Moreover, CSEs decreased proliferating cell nuclear antigen (PCNA) levels in JEG-3 cells in Western blot. Increased migration or invasion ability of JEG-3 cells following CSE treatment was also confirmed by a scratch or fibronectin invasion assay in vitro. Additionally, protein levels of E-cadherin as an epithelial maker were down-regulated, while the mesenchymal markers N-cadherin, snail and slug were up-regulated in a time-dependent manner. The metastasis marker, cathepsin D, was also down-regulated by CSE. Finally, CSEs significantly reduced the expression of hCG-β protein in JEG-3 cells. Overall, these results indicate that exposure of placental cells to CSE deregulates the cell cycle by altering the expression of cell cycle-related proteins and stimulates cell metastatic ability by altering EMT markers and cathepsin D expression. CSE exposure may also decrease hCG-β production as an endocrine marker, implying that cigarette smoke has adverse effects during pregnancy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Vacuolar protein sorting (VPS) genes encode a class of proteins involved in vesicular trafficking. Growing evidence suggests that VPS proteins play roles in tumor biology. Vacuolar protein sorting 52 (VPS52) is involved in retrograde transport of endosomes, and its roles in cancers have not been explored. This study investigated the genetic alterations, protein changes, biological role, and molecular mechanism of VPS52 in gastric cancer. Loss of heterozygosity of VPS52 was detected in 52.9% (9/17) of gastric cancer samples. Twenty-five percent (5/20) gastric cancer samples contained somatic stop-gain mutation of VPS52, two of which also had simultaneous loss of heterozygosity. Lack of VPS52 protein expression in gastric cancer tissue was found compared with pericancerous tissue and was significantly correlated with more advanced TNM staging and shorter 3-year overall survival. Overexpression of VPS52 significantly reduced viability and increased apoptosis in gastric cancer cells in vitro and reduced tumor volume and tumor weight in xenograft model in vivo. Activation of the cathepsin D/Bax/cytochrome C/caspase 9/caspase 3 pathway was detected in gastric cancer cells overexpressing VPS52. Collectively, VPS52 is a tumor suppressor gene in gastric cancer and could be used as a biomarker. VPS52 adenovirus could be a novel anti-tumor reagent for future gene therapy., Key Messages: Loss of heterozygosity and stop-gain mutation of VPS52 were found in gastric cancer. Negative expression of VPS52 significantly correlated with poor prognosis. VPS52 inhibited viability and induced apoptosis of gastric cancer cells in vitro. VPS52 reduced tumor volume and tumor weight in vivo. VPS52 activated the apoptotic pathway through cathepsin D in gastric cancer cells.

Ascertaining the vital origin of skin wounds is one of the most challenging problems in forensic pathology. The forensic literature describes biomarkers and methods for differentiating vital and postmortem wounds, although no clear conclusions have been reached. The aim of this study was to characterize human vital wounds by analyzing the concentrations of metallic ions and the expression of P-selectin and cathepsin D in skin wounds in the ligature marks in a cohort of suicidal hangings for which vitality was previously demonstrated.A total of 71 skin wounds were analyzed within a postmortem interval of 19 to 36 hours. The concentration of Fe, Zn, Mg, and Ca and the expression of P-selectin and cathepsin D were analyzed together and separately. The majority of autopsied suicidal hangings were men (86%) with complete hanging mode (60.7%) in which there was a high frequency of subcutaneous injuries (78.3%). High concentrations of Ca and Mg compared with Fe and Zn were found. Ca and Zn concentrations decreased, and Fe concentration increased with the seriousness of the injury. A high percentage of moderately negative expression of both proteins was correlated with subcutaneous injury and low or medium concentrations of Fe.In conclusion, the joint study of metallic ions and proteins allows to characterize and to differentiate an injured vital wound of noninjured skin, especially when the damage in the tissue affects to the majority of the structures of the skin, but these results will need to be complemented with other biomarkers in time-controlled samples to further help in the differentiation of vital and postmortem wounds.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

The nonreceptor kinase Janus kinase 2 (JAK2) has garnered attention as a promising therapeutic target for the treatment of CKD. However, being ubiquitously expressed in the adult, JAK2 is also likely to be necessary for normal organ function. Here, we investigated the phenotypic effects of JAK2 deficiency. Mice in which JAK2 had been deleted from podocytes exhibited an elevation in urine albumin excretion that was accompanied by increased podocyte autophagosome fractional volume and p62 aggregation, which are indicative of impaired autophagy completion. In cultured podocytes, knockdown of JAK2 similarly impaired autophagy and led to downregulation in the expression of lysosomal genes and decreased activity of the lysosomal enzyme, cathepsin D. Because transcription factor EB (TFEB) has recently emerged as a master regulator of autophagosome-lysosome function, controlling the expression of several of the genes downregulated by JAK2 knockdown, we questioned whether TFEB is regulated by JAK2. In immortalized mouse podocytes, JAK2 knockdown decreased TFEB promoter activity, expression, and nuclear localization. In silico analysis and chromatin immunoprecipitation assays revealed that the downstream mediator of JAK2 signaling STAT1 binds to the TFEB promoter. Finally, overexpression of TFEB in JAK2-deficient podocytes reversed lysosomal dysfunction and restored albumin permselectivity. Collectively, these observations highlight the homeostatic actions of JAK2 in podocytes and the importance of TFEB to autophagosome-lysosome function in these cells. These results also raise the possibility that therapeutically modulating TFEB activity may improve podocyte health in glomerular disease., (Copyright © 2017 by the American Society of Nephrology.)

Malate synthase is a condensing enzyme responsible for conversion of glyoxylate to malate in the presence of acetyl-CoA. This reaction helps in bypassing the TCA cycle reactions involving carbon loss and leads to diverting some of the carbon skeletons to gluconeogenic events while rest can continue to provide TCA cycle intermediates. Malate synthase (GlcB) is encoded by MRA&#95;1848 of Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a knockdown (KD) Mtb-Ra strain by down-regulating GlcB. The survival studies suggested increased susceptibility to oxidative and nitrosative stress as well as to rifampicin. The susceptibility profile was reversed in the presence of free radical scavengers. Also, KD showed reduced biofilm maturation, failed to enter persistent state, and showed reduced growth inside macrophages. The study of post-endocytosis events showed differences in late stage endosomal maturation behavior in macrophages infected with KD compared to WT. Increased iNOS, LAMP1 and cathepsin D expression was observed in macrophages infected with KD compared to WT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cathepsin D (Cath D) is overexpressed and secreted in a number of solid tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Inhibition of Cath D is regarded as an attractive pathway for the development of novel anticancer drugs. Our previous studies revealed that tasiamide B, a cyanobacterial peptide that contained a statine-like unit, exhibited good inhibition against Cath D and other aspartic proteases. Using this natural product as prototype, we designed and synthesized three new analogs, which bear isophthalic acid fragment at the N-terminus and isobutyl amine (1), cyclopropyl amine (2), or 3-methoxybenzyl amine (3) moiety at the C-terminus. Enzymatic assays revealed that all these three compounds showed moderate-to-good inhibition against Cath D, with IC 50 s of 15, 884, and 353 nM, respectively. Notably, compound 1 showed extreme selectivity for Cath D with 576-fold over Cath E and 554-fold over BACE1, which could be a valuable template for the design of highly potent and selective Cath D inhibitors. Additionally, compound 1 showed moderated activity against HeLa cell lines with IC 50 of 41.8 μM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Background: Oenothera biennis L., commonly known as evening primrose, harbours the flavonoids, steroids, tannins, fatty acids and terpenoids responsible for a diverse range of biological activity, such as antitumour, anti-arthritic and anti-inflammatory effects. In addition to the previous reports from aerial parts of this plant, studies related to antiproliferative or antimicrobial activity from the roots are warranted., Objective: To investigate antiproliferative and antimicrobial activity of compounds/mixture (1-8) isolated and characterized from the roots of O. biennis L. A possible mechanism of antiproliferative activity was also studied by targeting ornithine decarboxylase (ODC) and cathepsin D (CATD)., Study Design: Antiproliferative efficacy of the compounds/mixture was examined in selected cancer cell lines along with their probable mechanism of action. The antimicrobial activity was also studied against selected microbes (bacteria and fungi)., Methods: Antiproliferative potential was evaluated by MTT assay against selected cell lines. The mechanism of action was studied spectrophotometrically by targeting ODC and CATD using both an in-vitro and an in-silico approach. The antimicrobial efficiency was analysed using the disc diffusion and broth dilution methods., Key Findings: Oenotheralanosterol B (3) and the mixture of oenotheralanosterol A and oenotheralanosterol B (4) exhibited antiproliferative activity against breast, hepatic, prostate and leukaemia cancer cell lines as well as in mouse macrophages (IC 50 8.35-49.69 μg/ml). Oenotheralanosterol B (3) and the mixture of oenotheralanosterol A and oenotheralanosterol B (4) displayed a strong molecular interaction with succinate dehydrogenase (binding energy -6.23 and -6.84 kcal/mol and Ki 27.03 and 9.6 μm, respectively). Oenotheralanosterol A (1), oenotheralanosterol B (3) and mixture of oenotheralanosterol A and oenotheralanosterol B (4) potently inhibited the ODC activity with IC 50 ranging from 4.65 ± 0.35 to 19.06 ± 4.16 μg/ml and also showed a strong interaction with ODC (BE -4.17 to -4.46 kcal/mol). Oenotheralanosterol A (1), cetoleilyl diglucoside (2), oenotheralanosterol B (3), dihydroxyprenylxanthone acetylated (6) and dihydroxyprenylxanthone (7) inhibited CATD activity (IC 50 3.95 ± 0.49 to 24.35 ± 2.89 μg/ml). The in-silico molecular interaction analysis of compounds with CATD revealed the non-specific interaction. A moderate antimicrobial activity was observed against selected microbes with a growth inhibition ranging from 6 to 14 mm and minimum inhibitory concentration between 125 and 500 μg/ml. Oenotheralanosterol B (3) and dihydroxyprenylxanthone acetylated (6) exhibited better antimicrobial activity with an MIC range from 62.50 to 500 μg/ml., Conclusion: Oenotheralanosterol B (3) exhibited stronger antiproliferative and antimicrobial potential with respect to the other compounds tested, whereas oenotheralanosterol A (1) was a potent inhibitor of ODC and CATD. Hence, it is suggested that these in-vitro findings could be studied further in vivo for biological activity, safety evaluation and derivatization to enhance potency and efficacy., (© 2017 Royal Pharmaceutical Society.)

Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity., (© The Author 2017. Published by Oxford University Press.)

Oligomeric α-synuclein is a key mediator in the pathogenesis of Parkinson's disease (PD) and is mainly cleared by autophagy-lysosomal pathway, whose dysfunction results in the accumulation and cell-to-cell transmission of α-synuclein. In this study, concomitant with the accumulation of iron and oligomeric α-synuclein, higher expression of a lysosomal iron transporter, natural resistance-associated macrophage protein-1 (Nramp1), was observed in microglia in post-mortem striatum of sporadic PD patients. Using Nramp1-deficient macrophage (RAW264.7) and microglial (BV-2) cells as in-vitro models, iron exposure significantly reduced the degradation rate of the administered human α-synuclein oligomers, which can be restored by the expression of the wild-type, but not mutant (D543N), Nramp1. Likewise, under iron overload condition, mice with functional Nramp1 (DBA/2 and C57BL/6 congenic mice carrying functional Nramp1) had a better ability to degrade infused human α-synuclein oligomers than mice with nonfunctional Nramp1 (C57BL/6) in the brain and microglia. The interplay between iron and Nramp1 exhibited parallel effects on the clearance of α-synuclein and the activity of lysosomal cathepsin D in vitro and in vivo. Collectively, these findings suggest that the function of Nramp1 contributes to microglial degradation of oligomeric α-synuclein under iron overload condition and may be implicated in the pathogenesis of PD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Several lines of evidence indicate that adverse experience in early life may be a triggering factor for disturbances in the brain mitochondrial proteins and lead to the development of depression in adulthood. On the other hand, little is known about the impact of chronic administration of various antidepressant drugs on the brain mitochondria, as a target for the pharmacotherapy of depression. The purpose of our study was to compare the impact of chronic treatment with two antidepressant drugs with different mechanisms of action, a tricyclic antidepressant (TCA), imipramine, and an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class, fluoxetine, on the mitochondria-enriched subproteome profile in the hippocampus of 3-month-old male rats following a prenatal stress procedure (an animal model of depression). We clearly confirmed that chronic imipramine and fluoxetine administration not only normalized depression-like disturbances evoked by the prenatal stress procedure but also modulated the mitochondria-enriched subproteome profile in the hippocampus of adult offspring rats. In line with this, two-dimensional electrophoresis coupled with mass spectrometry showed a statistically significant down-regulation of 14-3-3 and cytochrome bc1 proteins and an up-regulation of COP9 signalosome expression after chronic imipramine treatment in the hippocampus of prenatally stressed offspring. Fluoxetine administration strongly up-regulated the expression of cathepsin D, one of the key proteins involved in the prevention of the development of neurodegenerative processes. Furthermore, this antidepressant treatment enhanced expression of proteins engaged in the improvement of learning and memory processes (STMN1, Dnm-1) as well as in mitochondrial biogenesis and defense against oxidative stress (DJ-1). These findings provide new evidence that chronic administration of antidepressants exerts a varied impact on the mitochondria-enriched subproteome in the hippocampus of adult rats following a prenatal stress procedure. In particular, the effect of fluoxetine requires additional experiments to elucidate the possible beneficial biological consequences underlying the effects mediated by this antidepressant., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Lysosomal dysfunction is implicated in human heart failure for which ischemic heart disease is the leading cause. Altered myocardial expression of CTSD (cathepsin D), a major lysosomal protease, was observed in human heart failure, but its pathophysiological significance has not been determined., Methods and Results: Western blot analyses revealed an increase in the precursor but not the mature form of CTSD in myocardial samples from explanted human failing hearts with ischemic heart disease, which is recapitulated in chronic myocardial infarction produced via coronary artery ligation in Ctsd +/+ but not Ctsd +/- mice. Mice deficient of Ctsd displayed impaired myocardial autophagosome removal, reduced autophagic flux, and restrictive cardiomyopathy. After induction of myocardial infarction, weekly serial echocardiography detected earlier occurrence of left ventricle chamber dilatation, greater decreases in ejection fraction and fractional shortening, and lesser wall thickening throughout the first 4 weeks; pressure-volume relationship analyses at 4 weeks revealed greater decreases in systolic and diastolic functions, stroke work, stroke volume, and cardiac output; greater increases in the ventricular weight to body weight and the lung weight to body weight ratios and larger scar size were also detected in Ctsd +/- mice compared with Ctsd +/+ mice. Significant increases of myocardial autophagic flux detected at 1 and 4 weeks after induction of myocardial infarction in the Ctsd +/+ mice were diminished in the Ctsd +/- mice., Conclusions: Myocardial CTSD upregulation induced by myocardial infarction protects against cardiac remodeling and malfunction, which is at least in part through promoting myocardial autophagic flux., (© 2017 American Heart Association, Inc.)

Neurologic phenotypes of cathepsin D (CTSD)-deficient mice, a murine model of neuronal ceroid lipofuscinoses, indicate the importance of CTSD for the maintenance of metabolism in central nervous system neurons. To further understand the role of CTSD in central nervous system neurons, we generated mice with a CTSD deficiency specifically in the Purkinje cells (PCs) (CTSD Flox/Flox ;GRID2-Cre) and compared their phenotypes with those of PC-selective Atg7-deficient (Atg7 Flox/Flox ;GRID2-Cre) mice. In both strains of mice, PCs underwent degeneration, but the CTSD-deficient PCs disappeared more rapidly than their Atg7-deficient counterparts. When CTSD-deficient PCs died, the neuronal cell bodies became shrunken, filled with autophagosomes and autolysosomes, and had nuclei with dispersed small chromatin fragments. The dying Atg7-deficient PCs also showed similar ultrastructures, indicating that the neuronal cell death of CTSD- and Atg7-deficient PCs was distinct from apoptosis. Immunohistochemical observations showed the formation of calbindin-positive axonal spheroids and the swelling of vesicular GABA transporter-positive presynaptic terminals that were more pronounced in Atg7-deficient PCs than in CTSD-deficient PCs. An accumulation of tubular vesicles may have derived from the smooth endoplasmic reticulum; nascent autophagosome-like structures with double membranes was a common feature in the swollen axons of these PCs. These results suggested that PCs were more vulnerable to CTSD deficiency in lysosomes than to autophagy impairment, and this vulnerability does not depend on the severity of axonal swelling., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Autophagy and lysosomal function are important for protein homeostasis and their dysfunction have been associated with Alzheimer's disease (AD). Increased immunoreactivities of an important lysosomal protease, cathepsin D (Cat D), are evident in amyloid plaques and neurons in patients with AD. This study tests the hypothesis that deleting one allele of the cathepsin D gene (Ctsd) impacts cerebral β-amyloidosis in amyloid-β precursor protein (APP)sw/PS1dE9 (APP/PS1) double transgenic mice. Despite a significant 38% decrease in Cat D level in APP/PS1/Ctsd+/- compared with APP/PS1/Ctsd+/+ mice, no changes in steady state levels and deposition of Aβ were found in the brain. There were also no differences in APP processing, the levels of two other Aβ-degrading proteases, the levels of autophagy related protein, such as LAMP2, P62, LC3-I, LC3-II, and Beclin-1, or the markers of neuroinflammation, observed between the APP/PS1/Ctsd+/+ and APP/PS1/Ctsd+/- mice. Our findings demonstrate that in wild-type mice, Cat D protein levels are either in excess or redundant with other factors in the brain, and at least one allele of Ctsd is dispensable for cerebral β-amyloidosis and autophagy in APP/PS1 transgenic mice., (© 2017 International Society for Neurochemistry.)

Cathepsin D is reportedly to be closely associated with tumor development, migration, and invasion, but its pathological mechanism is not fully elucidated. We aimed to evaluate phenotypic changes and molecular events in response to cathepsin D knockdown. Lowering endogenous cathepsin D abundance (CR) induced senescence in HeLa cells, leading to reduced rate of cell proliferation and impaired tumorigenesis in a mouse model. Quantitative proteomics revealed that compared with control cells (EV), the abundances of several typical lysosomal proteases were decreased in the lysosomal fraction in CR cells. We further showed that cathepsin D knockdown caused increased permeability of lysosomal membrane and reactive oxygen species accumulation in CR cells, and the scavenging of reactive oxygen species by antioxidant was able to rescue cell senescence. Despite the increased reactive oxygen species, the proteomic data suggested a global reduction of redox-related proteins in CR cells. Subsequent analysis indicated that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2), which regulates the expression of groups of antioxidant enzymes, was down-regulated by cathepsin D knockdown. Importantly, Nrf2 overexpression significantly reduced cell senescence. Although transient oxidative stress promoted the accumulation of Nrf2 in the nucleus, we showed that the Nrf2 protein exited nucleus if oxidative stress persisted. In addition, when cathepsin D was transiently knocked down, the cathepsin-related events followed a sequential order, including lysosomal leakage during the early stage, followed by oxidative stress augmentation, and ultimately Nrf2 down-regulation and senescence. Our results suggest the roles of cathepsin D in cancer cells in maintaining lysosomal integrity, redox balance, and Nrf2 activity, thus promoting tumorigenesis. The MS Data are available via ProteomeXchange with identifier PXD002844., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In insects, proteolytic enzymes are involved in food digestion and the metamorphosis process. In the present study, the full-length cDNA of an aspartic proteinase, Spodoptera exigua cathepsin D (SeCatD), was cloned, and its functions in metamorphosis were characterized. SeCatD contains an open reading frame of 1152 nucleotides, encoding a 384-amino acid polypeptide including a signal peptide and two functional domains (family A1 propeptide of amino acids (19-45) and a cathepsin D-like domain of 327 amino acids (55-381)). Three-dimensional structure analysis indicated that Asp66 and Asp251 may play important role in hydrolysis. Recombinant SeCatD was expressed in Sf9 insect cells and verified via SDS-PAGE and Western blot, the molecular mass of the expressed SeCatD was approximately 42kDa. The enzyme had an optimal pH value of 3 for activity. In addition, the tissue expression profile of SeCatD during metamorphosis was obtained, and the data demonstrated that SeCatD was expressed increasingly in the fat body and midgut, but not in the epidermis. Finally, injection of dsRNA-SeCatD into the fifth-instar larvae significantly reduced SeCatD expression and larvae survival rate compared to a dsRNA-GFP treatment. These data imply that SeCatD may function during metamorphosis and may represent a target for insect control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism., Conclusion: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

BACE1 is a major therapeutic target for prevention and treatment of Alzheimer's disease. Developing inhibitors that can selectively target BACE1 in favor of other proteases, especially cathepsin D (CatD), has presented significant challenges. Here, we investigate the conformational dynamics and protonation states of BACE1 and CatD using continuous constant pH molecular dynamics with pH replica-exchange sampling protocol. Despite similar structure, BACE1 and CatD exhibit markedly different active site dynamics. BACE1 displays pH-dependent flap dynamics that controls substrate accessibility, while the CatD flap is relatively rigid and remains open in the pH range 2.5-6. Interestingly, although each protease hydrolyzes peptide bonds, the protonation states of the catalytic dyads are different within the active pH range. The acidic and basic components of the BACE1 catalytic dyad are clear, while either aspartic acid of the CatD catalytic dyad could play the role of acid or base. Finally, we investigate binding of the inhibitor LY2811376 developed by Eli Lilly to BACE1 and CatD. Surprisingly, in the enzyme active pH range, LY2811376 forms a stronger salt bridge with the catalytic dyad in CatD than in BACE1, which might explain the retinal toxicity of the inhibitor related to off-target inhibition of CatD. This work highlights the complexity and challenge in structure-based drug design where receptor-ligand binding induces protonation state change in both the protein and the inhibitor. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Studies in animal models of type 2 diabetes have shown that glucagon-like peptide 1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key lysosomal-mediated process of autophagy is unknown. In this study, we report that treatment of INS-1E β-cells and primary islets with glucolipotoxicity (0.5 mmol/L palmitate and 25 mmol/L glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilization and release of cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Cotreatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. Small interfering RNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4. Collectively, our data highlight lysosomal dysfunction as a critical mediator of β-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy/lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signaling pathway as a potential focus., (© 2017 by the American Diabetes Association.)

Activity of cathepsin D and phagocytosis of macrophages from vaginal lavage fluid, peritoneal exudation, and spleen were studied in mice of sensitive (DBA/2) and resistant (BALB/c) lines after intravaginal infection with type 2 herpes simplex virus and vaccination. Activity of cathepsin D and intensity of phagocytosis (irrespective of the macrophage source) and their ratio in BALB/c mice in early terms after infection were close to the control levels taken as a unit. In DBA/2 mice, these parameters and their balance were shifted and changes in cathepsin D activity depended on the time after challenge. Activities of cellular and extracellular cathepsin D increased sharply on day 1 postinfection under conditions of local virus interaction with the vaginal mucosa and activation of the pathological process. Later, after generalization of the infection, activity of cathepsin D decreased, while phagocytosis increased in all the studied macrophage populations. Vaccination corrected the cathepsin D/phagocytosis imbalance and created conditions for rapid elimination of the virus.

Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle., (Copyright © 2017 Elsevier Inc. All rights reserved.)