miR-185-5p Represses Cells Growth and Metastasis of Osteosarcoma via Targeting Cathepsin E.

Osteosarcoma (OS) is a malignant bone tumor characterized by poor prognosis due to its regional invasion and early metastasis. In this study, we aimed to find the role and the underlying mechanism of Cathepsin E (CTSE) in OS growth and metastasis. We found CTSE is upregulated in metastatic OS, rather than in the primary lesion, as confirmed by RT-qPCR and western blot analysis of clinical OS samples. Furthermore, both in vitro and in vivo experiments illustrated that CTSE promoted both growth and metastasis of OS cells, partially mediated through the modulation of Epithelial-Mesenchymal Transition (EMT). Bioinformatics analysis predicted that miR-185-5p downregulates CTSE via directly binding to the 3'UTR of CTSE, which was verified by luciferase reporter assay and rescue assays. This study reported for the first time that CTSE is a potential biomarker in OS tumorigenesis and metastasis, providing a promising therapeutic target for OS treatment.

Microglial cathepsin E plays a role in neuroinflammation and amyloid ß production in Alzheimer's disease.

Regulation of neuroinflammation and ß-amyloid (Aß) production are critical factors in the pathogenesis of Alzheimer's disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock-in mice, called APPNL-G-F mice, significantly reduced Aß accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF-related apoptosis-inducing ligand, which plays an important role in microglia-mediated NF-κB-dependent neuroinflammation and neuronal Aß production by beta-site APP cleaving enzyme 1. Furthermore, cannula-delivered CatE inhibitors improved memory function and reduced Aß accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aß pathology.

Grassystatin-derived peptides selectively inhibit cathepsin E and have low affinity to cathepsin D.

Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research.

Glycoprotein 5 Is Cleaved by Cathepsin E during Porcine Reproductive and Respiratory Syndrome Virus Membrane Fusion.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

First report of cathepsin E in a teleost (Korean rose bitterling, Rhodeus uyekii): Molecular characterisation and tissue distribution.

We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.

Cathepsin E expression and activity: Role in the detection and treatment of pancreatic cancer.

Cathepsin E (CTSE) is an intracellular, hydrolytic aspartic protease found to be expressed in cells of the immune and gastrointestinal systems, lymphoid tissues, erythrocytes, and cancer cells. The precise functions are not fully understood; however, various studies have investigated its numerous cell-type specific roles. CTSE expression has been shown to be a potential early biomarker for pancreatic ductal adenocarcinoma (PDAC). PDAC patients have low survival rates mostly due to the lack of early detection methods. CTSE-specific activity probes have been developed and tested to assist in tumor imaging and functional studies investigating the role of CTSE expression in PDAC tumors. Furthermore, a CTSE protease-specific, photodynamic therapy pro-drug was developed to explore its potential use to treat tumors that express CTSE. Since CTSE is expressed in pancreatic diseases that are risk factors for PDAC, such as pancreatic cysts and chronic pancreatitis, learning about its function in these disease types could assist in early PDAC detection and in understanding the biology of PDAC progression. Overall, CTSE expression and activity shows potential to detect PDAC and other pancreatic diseases. Further research is needed to fully understand its functions and potential translational applicability.

Defining Genetic Variation in Widely Used Congenic and Backcrossed Mouse Models Reveals Varied Regulation of Genes Important for Immune Responses.

Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.

Oestrogen regulates the expression of cathepsin E-A-like gene through ERΒ in liver of chicken (Gallus gallus).

The cathepsin E-A-like, also known as 'similar to nothepsin', is a new member of the aspartic protease family, which may take part in processing of egg yolk macromolecules, due to it was identified in the chicken egg-yolk. Previously, studies have suggested that the expression of cathepsin E-A-like increased gradually during sexual maturation of pullets, but the exact regulation mechanism is poorly understood. In this study, to gain insight into the function and regulation mechanism of the gene in egg-laying hen, we cloned the cathepsin E-A-like gene and evaluated its evolutionary origin by using both phylogenetic and syntenic methods. The mode of the gene expression regulation was analysed through stimulating juvenile hens with 17ß-estradiol and chicken embryo hepatocytes with 17ß-estradiol combined with oestrogen receptor antagonists including MPP, ICI 182,780 and tamoxifen. Our results showed that cathepsin E-A-like was an orthologoues gene with nothepsin, which is present in birds but not in mammals. The expression of cathepsin E-A-like significantly increased in a dose-dependent manner after the juvenile hens were treated with 17ß-estradiol (P < 0.05). Compared with the 17ß-estradiol treatment group, the expression of cathepsin E-A-like was not significantly changed when the hepatocytes were treated with 17ß-estradiol combined with MPP (P < 0.05). In contrast, compared with the 17ß-estradiol combined with MPP treatment group, the expression of cathepsin E-A-like was significantly downregulated when the hepatocytes were treated with 17ß-estradiol combined with tamoxifen or ICI 182,780 (P < 0.05). These results demonstrated that cathepsin E-A-like shared the same evolutionary origin with nothepsin. The expression of cathepsin E-A-like was regulated by oestrogen, and the regulative effect was predominantly mediated through ER-Β in liver of chicken.

PPARα-Target Gene Expression Requires TIS21/BTG2 Gene in Liver of the C57BL/6 Mice under Fasting Condition.

The TIS21/BTG2/PC3 gene belongs to the antiproliferative gene (APRO) family and exhibits tumor suppressive activity. However, here we report that TIS21 controls lipid metabolism, rather than cell proliferation, under fasting condition. Using microarray analysis, whole gene expression changes were investigated in liver of TIS21 knockout (TIS21-KO) mice after 20 h fasting and compared with wild type (WT). Peroxisome proliferator-activated receptor alpha (PPARα) target gene expression was almost absent in contrast to increased lipid synthesis in the TIS21-KO mice compared to WT mice. Immunohistochemistry with hematoxylin and eosin staining revealed that lipid deposition was focal in the TIS21-KO liver as opposed to the diffuse and homogeneous pattern in the WT liver after 24 h starvation. In addition, cathepsin E expression was over 10 times higher in the TIS21-KO liver than that in the WT, as opposed to the significant reduction of thioltransferase in both adult and fetal livers. At present, we cannot account for the role of cathepsin E. However, downregulation of glutaredoxin 2 thioltransferase expression might affect hypoxic damage in the TIS21-KO liver. We suggest that the TIS21/BTG2 gene might be essential to maintain energy metabolism and reducing power in the liver under fasting condition.

Peptide aptamer-modified single-walled carbon nanotube-based transistors for high-performance biosensors.

Biosensors employing single-walled carbon nanotube field-effect transistors (SWCNT FETs) offer ultimate sensitivity. However, besides the sensitivity, a high selectivity is critically important to distinguish the true signal from interference signals in a non-controlled environment. This work presents the first demonstration of the successful integration of a novel peptide aptamer with a liquid-gated SWCNT FET to achieve highly sensitive and specific detection of Cathepsin E (CatE), a useful prognostic biomarker for cancer diagnosis. Novel peptide aptamers that specifically recognize CatE are engineered by systemic in vitro evolution. The SWCNTs were firstly grown using the thermal chemical vapor deposition (CVD) method and then were employed as a channel to fabricate a SWCNT FET device. Next, the SWCNTs were functionalized by noncovalent immobilization of the peptide aptamer using 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker. The resulting FET sensors exhibited a high selectivity (no response to bovine serum albumin and cathepsin K) and label-free detection of CatE at unprecedentedly low concentrations in both phosphate-buffered saline (2.3 pM) and human serum (0.23 nM). Our results highlight the use of peptide aptamer-modified SWCNT FET sensors as a promising platform for near-patient testing and point-of-care testing applications.

Combination of active targeting, enzyme-triggered release and fluorescent dye into gold nanoclusters for endomicroscopy-guided photothermal/photodynamic therapy to pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating malignancies in patients, and there is an urgent need for an effective treatment method. Herein, we report a novel gold nanocluster-based platform for confocal laser endomicroscopy-guided photothermal therapy (PTT)/photodynamic therapy (PDT) for PDAC, which consists of four components: the PTT-carrier gold nanocluster, an active targeting ligand U11 peptide, a Cathepsin E (CTSE)-sensitive PDT therapy prodrug, and a CTSE-sensitive imaging agent (cyanine dye Cy5.5). Due to the strong coupling among cross-linked gold nanoparticles (AuNPs), the surface plasmon resonance peak of nanoclusters shifts to the near-infrared (NIR) region, thus making the nanoclusters useful in the effective PTT therapy. In the system, the labeling of nanoclusters with U11 peptide can distinctly increase their affinity and accelerate their uptake by pancreatic cancer cells. Cell apoptosis staining demonstrates that, upon incorporation of the uPAR-targeted unit, the antitumor efficacy of CTSE-sensitive nanocluster AuS-U11 is significantly enhanced with respect to that of the non-targeted nanocluster AuS-PEG and the insensitive nanocluster AuC-PEG. In vivo and ex vivo optical imaging confirms the high accumulation of AuS-U11 in the in situ pancreatic tumor model. Therapeutic studies further show that the combination of active targeting for tumor tissue, enzyme-triggered drug release of 5-ALA and fluorescent dye Cy5.5 in nanoclusters AuS-U11 could achieve optimal therapeutic efficacy with endomicroscopy-guided photothermal/photodynamic therapy with minimal side effects. As a consequence, the delicate gold nanocluster concept provides a promising strategy to enhance the therapy efficiency in the most challenging PDAC treatment.

Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts.

Purpose: Pancreatic cysts are estimated to be present in 2%-3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts.Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 µL of cyst fluid.Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity.Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865-74. ©2017 AACR.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine.

This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency-high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 µm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology.

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors.

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

The Critical Role of Proteolytic Relay through Cathepsins B and E in the Phenotypic Change of Microglia/Macrophage.

Proteinase cascades are part of the basic machinery of neuronal death pathways. Neuronal cathepsin B (CatB), a typical cysteine lysosomal protease, plays a critical role in neuronal death through lysosomal leakage or excessive autophagy. On the other hand, much attention has been paid to microglial CatB in neuronal death. We herein show the critical role of proteolytic relay through microglial CatB and CatE in the polarization of microglia/macrophages in the neurotoxic phenotype, leading to hypoxia/ischemia (HI)-induced hippocampal neuronal damage in neonatal mice. HI caused extensive brain injury in neonatal wild-type mice, but not in CatB(-/-) mice. Furthermore, HI-induced polarization of microglia/macrophages in the neurotoxic phenotype followed by the neuroprotective phenotype in wild-type mice. On the other hand, microglia/macrophages exhibited only the early and transient polarization in the neuroprotective phenotype in CatB(-/-) mice. CA-074Me, a specific CatB inhibitor, significantly inhibited the neuronal death of primary cultured hippocampal neurons induced by the conditioned medium from cultured microglia polarized in the neurotoxic phenotype. Furthermore, CA-074Me prevented the activation of nuclear factor-κB (NF-κB) in cultured microglia by inhibiting autophagic inhibitor of κBα degradation following exposure to oxygen-glucose deprivation. Rather surprisingly, CatE increased the CatB expression after HI by the liberation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from microglia through the proteasomal pathway. A significant increase in CatB and CatE levels was found exclusively in microglia/macrophages after HI. Thus, a proteolytic relay through the early CatE/TRAIL-dependent proteosomal and late CatB-dependent autophagic pathways for NF-κB activation may play a critical role in the polarization of microglia/macrophages in the neurotoxic phenotype. Significance statement: Proteinase cascades are part of the basic machinery of neuronal death pathways. Cathepsin B, a typical cysteine lysosomal protease, plays a critical role in neuronal death through lysosomal leakage or excessive autophagy in neurons. On the other hand, much attention has been also paid to the role of microglial cathepsin B in neuronal death. In this study, using in vivo and in vitro models of relevance to brain ischemia, we found a critical role of proteolytic relay through cathepsin B and cathepsin E in the neurotoxic polarization of microglia/macrophages, which is responsible for aggravation of hypoxia/ischemia-induced neuronal injury. These findings suggest orally active selective inhibitors of cathepsin B or cathepsin E as promising pharmacological agents for the treatment of ischemic brain injury.

Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.

BACKGROUND: Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. METHODS: BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. RESULTS: The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. CONCLUSIONS: Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.

Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes.