Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.     

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.     

Proteomic profiling of acrolein adducts in human lung epithelial cells

Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology.     

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

Background  

The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive.     

Elastinolytic activity of human cathepsin L.

The hydrolysis of a tritiated elastin substrate by the human cysteine proteinases cathepsins B and L has been studied. Cathepsin L was found to be at least 100-fold more active on this substrate than cathepsin B. The specific activity of cathepsin L at pH 5.5 for hydrolysis of elastin was about the same as that of pig pancreatic elastase at its optimum pH of 8.8.     

Post-transcriptional regulation of human cathepsin L expression

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5'UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5'UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5'UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5'UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5'UTRs of human cathepsin L variants are involved in regulating its expression.     

Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells

Copper oxide nanoparticles (CuO NPs) are increasingly used in various applications. Recent studies suggest that oxidative stress may be the cause of the cytotoxicity of CuO NPs in mammalian cells. However, little is known about the genotoxicity of CuO NPs following exposure to human cells. This study was undertaken to investigate CuO NPs induced genotoxic response through p53 pathway in human pulmonary epithelial cells (A549). In addition, cytotoxicity and oxidative stress markers were also assessed. Results showed that cell viability was reduced by CuO NPs and degree of reduction was dose dependent. CuO NPs were also found to induce oxidative stress in dose-dependent manner indicated by depletion of glutathione and induction of lipid peroxidation, catalase and superoxide dismutase. The expression of Hsp70, the first tier biomarker of cellular damage was induced by CuO NPs. Further, CuO NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and MSH2 expression. These results demonstrate that CuO NPs possess a genotoxic potential in A549 cells which may be mediated through oxidative stress. Our short-term exposure study of high level induction of genotoxic response of CuO NPs will need to be further investigated to determine whether long-term exposure consequences may exist for CuO NPs application.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.     

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells

Dissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalational anthrax. We recently reported that B. anthracis could adhere to and be internalized by cultured intestinal epithelial and fibroblast cells. Here, using gentamicin protection assays and/or electron microscopy, we found that Sterne strain 7702 spores were able to adhere to and subsequently be internalized by polarized A549 cells and primary human small airway epithelial cells. We showed for the first time that internalized spores were able to survive and that spores could translocate across an A549 cell barrier from the apical side to the basolateral side without disrupting the barrier integrity, suggesting a transcellular route. In addition, dormant spores of fully virulent Ames and UT500 strains were able to adhere to A549 cells at a frequency similar to that of 7702, whereas the capsule in germinated Ames and UT500 spores prevented adherence. Fluorescence microscopy also revealed that dormant Ames spores were internalized at a frequency similar to that of 7702. These findings highlight the possibility of a novel route of dissemination in which B. anthracis utilizes epithelial cells of the lung. The implications of these results to B. anthracis pathogenesis are discussed.     

Curcumin modulates eukaryotic initiation factors in human lung adenocarcinoma epithelial cells

Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors (eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0–240 μM curcumin for 4–96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.     

Ultrasensitive internally quenched substrates of human cathepsin L

Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO₂)-NH₂ with high specificity constant (k_cat/K_M = 2.6 × 10⁷ M⁻¹ s⁻¹) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.     

Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.