A survey of volatile species in Oort cloud comets C/2001 Q4 (NEAT) and C/2002 T7 (LINEAR) at millimeter wavelengths

The line emission in the coma was measured in the comets C/2001 Q4 (NEAT) and C/2002 T7 (LINEAR), that were observed on five consecutive nights, 7-11 May 2004, at heliocentric distances of 1.0 and 0.7 AU, respectively, by means of high-resolution spectroscopy using the 10-m Submillimeter Telescope (SMT). We present a search for six parent- and product-volatile species (HCN, H2CO, CO, CS, CH3OH, and HNC) in both comets. Multiline observations of the CH3OH J = 5-4 series allow us to estimate the rotational temperature using the rotation diagram technique. We derive rotational temperatures of 54(9) K for C/2001 Q4 (NEAT) and 119(34) K for C/2002 T7 (LINEAR) that are roughly consistent with observations of other comets at similar distances from the Sun. The gas production rates of material are computed using a spherically symmetric molecular excitation code that includes collisions between neutrals and electrons. We find an HCN production rate of 2.96(5)e26 molec.s-1 for comet C/2001 Q4 (NEAT), corresponding to a mixing ratio with respect to H2O of 1.12(2)e-3. The mean HCN production rate during the observing period is 4.54(10)e26 molec.s-1 for comet C/2002 T7 (LINEAR), which gives a Q_HCN/Q_H2O mixing ratio of 1.51(3)e-3. With systematically lower mixing ratios in comet C/2001 Q4 (NEAT), production rate ratios of the observed species with respect to H2O lie within the typical ranges of dynamically new comets in both objects. We find a relative low abundance of CO in C/2001 Q4 (NEAT) compared to the observed range in other comets based on millimeter/submillimeter observations, and a significant upper limit on the CO production in C/2002 T7 (LINEAR) is derived. Depletion of CO suggests partial evaporation from the surface layers during previous visits to the outer Solar System and agrees with previous measurements of dynamically new comets.Comment: 20 pages, 18 figures. Minor changes to match the published versio

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRP ML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL

Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis

Running-Induced Systemic Cathepsin B Secretion Is Associated with Memory Function

Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. In vivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition

A Possible Contribution of Altered Cathepsin B Expression to the Development of Skin Sclerosis and Vasculopathy in Systemic Sclerosis

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc

The identification and characterisation of the causative gene mutation for keratolytic winter erythema (KWE) in South African families

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment for the degree of Doctor of Philosophy Johannesburg, 2017Keratolytic winter erythema (KWE) is a rare autosomal dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling, and symptoms worsen in winter. KWE is relatively common in South African (SA) Afrikaners and was mapped to 8p23.1-p22 through a common haplotype in SA families. The aim of this study was to identify and characterize the causal mutation for KWE in SA families. Targeted resequencing of 8p23.1-22 was performed in three families and seven unrelated controls. Reads were aligned to the reference genome using BWA. GATK and Pindel were used to call small and large structural variants, respectively. A 7.67 kb tandem duplication was identified upstream of the CTSB gene and encompassing an enhancer element that is active in a keratinocytes (based on H3K27ac data). The tandem duplication segregated completely with the KWE. The tandem duplication overlaps with a 15.93 kb tandem duplication identified in two Norwegian families at a 2.62 kb region encompassing the active enhancer suggesting that the duplication of the enhancer leads to the KWE phenotype. Existing chromatin structure, CTCF binding and chromatin interaction data from several cell lines, including keratinocytes were analysed and three potential topological subdomains were identified, all containing the enhancer and CTSB, or CTSB and FDFT1 or both genes and NEIL2. Additionally, we showed that the enhancer’s activity correlated with CTSB expression, but not with FDFT1 and NEIL2 expression in differentiating keratinocytes and other cell lines. RNA polymerase II ChIA-PET interaction data in cancer cell lines showed that the enhancer interacts with CTSB but not FDFT1 or NEIL2. These data suggest that the enhancer normally regulates CTSB expression. Relative gene expression and immunohistochemistry from palmar biopsies from South African and Norwegian participants (7 Affected and 7 Controls) showed a significantly higher expression of CTSB, but not FDFT1 and NEIL2, in affected individuals compared to the controls and that CTSB was significantly more abundant in the granular layer of affected individuals compared to controls. We conclude that the enhancer duplication causes KWE by upregulating CTSB expression and causing an overabundance of CTSB in the granular layer of the epidermis.MT201

Elucidating the role of cathepsin B in the lifecycle of influenza A virus

Influenza virus type A (IVA) is the etiologic agent responsible for the febrile respiratory illness referred to as the flu. Seasonal and occasionally pandemic IVA-associated illness is a significant cause of morbidity and mortality worldwide, and presents a significant burden to the healthcare system. Our previous work showed that the propagation of IVA required the lysosomal protease cathepsin B (CTSB), though the mechanism behind this dependency was not elucidated. This study further examined the role of CTSB by using CTSB-deficient (CTSB-/-) macrophages and the CTSB-specific chemical inhibitor CA-074 Me (CaMe) in human lung epithelial cells. CTSB-/- and CA-074 Me-treated cells showed no defect in either the uptake of virus particles, nor their replication following endosomal escape compared to wildtype or non-treated cells, respectively. However, CTSB-/- and CA-074 Me-treated cells had significantly less hemagglutinin (HA) protein both inside and on the surface of infected cells, as determined by both Western blotting and confocal immunofluorescence microscopy. These results suggest that CTSB is required for a step(s) in the viral lifecycle following entry into host cells, either before or during the synthesis of viral proteins, and possibly during the transport of viral components to the host membrane. Further work is necessary to determine the mechanistic details of these observations, and may yield novel potential therapies for influenza infections

Loss of Cathepsin B and L Leads to Lysosomal Dysfunction, NPC-Like Cholesterol Sequestration and Accumulation of the Key Alzheimer's Proteins

Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer’s proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases

Cathepsin b: a potential prognostic marker for inflammatory breast cancer

<p>Abstract</p> <p>Background</p> <p>Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC.</p> <p>Methods</p> <p>In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1), a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups.</p> <p>Results</p> <p>Our results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC.</p> <p>Conclusions</p> <p>CTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.</p