RESUMEN
The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Anticuerpos Monoclonales , Ratones Transgénicos , Acinetobacter baumannii/inmunología , Acinetobacter baumannii/genética , Animales , Humanos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Infecciones por Acinetobacter/inmunología , Infecciones por Acinetobacter/prevención & control , Infecciones por Acinetobacter/microbiología , Ratones , Antibacterianos/farmacología , Anticuerpos Antibacterianos/inmunología , Femenino , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana/inmunología , Farmacorresistencia Bacteriana/genética , Lipopolisacáridos/inmunologíaRESUMEN
Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors.
Asunto(s)
Neoplasias Pulmonares , Humanos , Catepsina L/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Células HeLa , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Microambiente TumoralRESUMEN
L-leucyl-leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft-vs-host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U-937 and THP-1 promonocytes as models for monocytic cells, U-87-MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C-dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C-the most upstream molecule in the pathway-in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.
Asunto(s)
Apoptosis , Catepsina C/metabolismo , Dipéptidos/farmacología , Inmunosupresores/farmacología , Monocitos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Monocitos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.
Asunto(s)
Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Membrana Celular/metabolismo , Neoplasias/metabolismo , Proteómica/métodos , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Movimiento Celular , Humanos , Macrófagos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Invasividad Neoplásica , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato , Proteínas ras/metabolismoRESUMEN
Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported. Although most of these approaches can identify up to thousands of substrate cleavage events in a single experiment, they are often time consuming and methodologically challenging as some of these approaches require rather complex sample preparation procedures. For such reasons their application is often limited to those labs that initially introduced them. Here, we report on a fast and simple approach for proteomic profiling of protease specificities (fast profiling of protease specificity (FPPS)), which can be applied to complex protein mixtures. FPPS is based on trideutero-acetylation of novel N-termini generated by the action of proteases and subsequent peptide fractionation on Stage Tips containing ion-exchange and reverse phase chromatographic resins. FPPS can be performed in 2 days and does not require extensive fractionation steps. Using this approach, we have determined the specificity profiles of the cysteine cathepsins K, L and S. We further validated our method by comparing the results with the specificity profiles obtained by the N-terminal combined fractional diagonal chromatography method. This comparison pointed to almost identical substrate specificities for all three cathepsins and confirmed the reliability of the FPPS approach. All MS data have been deposited in the ProteomeXchange with identifiers PXD001536 and PXD001553 (http://proteomecentral.proteomexchange.org/dataset/PXD001536; http://proteomecentral.proteomexchange.org/dataset/PXD001553).
Asunto(s)
Catepsina K/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Secuencia de Aminoácidos , Catepsina K/química , Catepsina L/química , Catepsinas/química , Línea Celular Tumoral , Cromatografía Liquida/métodos , Humanos , Péptidos/química , Péptidos/metabolismo , Proteómica/métodos , Especificidad por Sustrato , Espectrometría de Masas en Tándem/métodosRESUMEN
The potential role of cysteine cathepsins in tumor necrosis factor-related apoptosis-inducing ligand(TRAIL/Apo2L)- and CD95 (Fas/APO-1)-induced apoptosis was investigated using four different cell lines (HeLa, HuH-7, Jurkat, and U-937). All four cell lines exhibited different levels of cathepsins and responded differently to apoptosis triggering, with Jurkat cells being the most sensitive and the only ones that were sensitive to the agonistic anti-APO-1 antibody. Apoptosis was accompanied by caspase activation, loss of the mitochondria and lysosome integrity, and the release of cysteine cathepsins into the cytosol, as judged based on the hydrolysis of the cysteine cathepsin substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin and by the immunological detection of cathepsin B. The inhibition of caspases by the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis,including the mitochondrial and lysosomal membrane permeabilization, as well as cathepsin release into the cytosol, consistent with caspases playing a crucial role in the process. Conversely, however, although the broad-spectrum cysteine cathepsin inhibitor (2 S ,3 S )-trans -epoxysuccinyl-leucyl amido-3-methyl-butane ethyl ester and the more cathepsin B-selective inhibitor[(2 S ,3 S )-3-propylcarbamoyloxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester completely blocked cathepsin activity, these inhibitors neither prevented apoptosis including the mitochondrial and lysosomal membrane permeabilization, as well as cathepsin release into the cytosol, consistent with caspases playing a crucial role in the process. Conversely, however, although the broad-spectrum cysteine cathepsin inhibitor (2 S ,3 S )-trans -epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester and the more cathepsin B-selective inhibitor[(2 S ,3 S )-3-propylcarbamoyloxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester completely blocked cathepsin activity, these inhibitors neither prevented apoptosis and its progression nor the mitochondrial and lysosomal membrane permeabilization associated with this type of cell death. Consequently, cathepsin release into the cytosol was also not prevented. Together, these data indicate that cysteine cathepsins are not required for the TRAIL- and CD95-mediated apoptosis in various human cancer cell lines. This does not, however, rule out that lysosomes and cysteine cathepsins are involved in the amplification, but not in the initiation, of death receptor-mediated apoptosis in certain cell lines or under different stimulation conditions than the ones employed here.
Asunto(s)
Apoptosis , Cisteína Endopeptidasas/metabolismo , Neoplasias/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptor fas/metabolismo , Caspasas/metabolismo , Catepsina B/metabolismo , Línea Celular Tumoral , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias/patologíaRESUMEN
Sodium nitroprusside (SNP) is a nitric oxide (â¢NO) donor in vitro and in vivo. In this paper the time variation of the intracellular water proton nuclear magnetic resonance (NMR) effective relaxation time T'(2a) in SNP-treated human erythrocyte suspensions, containing 10 mM membrane impermeable paramagnetic MnCl2, has been measured. The observed T'(2a) time-course was analyzed in terms of the two mechanisms by which released â¢NO affects T'(2a). These are, respectively, enhancement of the intracellular water proton intrinsic NMR relaxation rate 1/T(2a) by paramagnetism of â¢NO subsequently bonded to iron atoms of intracellular deoxyhemoglobin, and suppression of diffusional water permeability P(d) as a consequence of nitrosylation of aquaporin-1 (AQP1) channel Cys189, either by direct reaction with â¢NO or with one of the â¢NO oxidation products, such as N2O3. The bound â¢NO on the Cys189 thiol residue appears to impose a less efficient barrier to water permeation through AQP1 than the larger carboxyphenylmercuryl residue from p-chloromercuribenzoate. The effect of â¢NO on P(d) is discussed in terms of NO-induced vasodilation.