To reveal whether the signal is lysosomal, LysoTracker (Life technologies, 50 nM) was added minutes before imaging. programmed cell death in healthy and disease says. Introduction Apoptosis (programmed cell death) is the major process by which multi-cellular organisms eliminate excessive, damaged and potentially dangerous cells. 1 Both normal and pathological processes such as embryonic development, malignancy, auto-immune disorders, ischemia and reperfusion or Parkinson’s and Alzheimer’s disease involve apoptotic cell death processes. Importantly, the induction of apoptosis in cancerous cells is the desired outcome of many cancer chemotherapeutic treatments and initiation or inhibition of apoptosis is usually a key element in numerous therapies.2,3 Therefore, the availability of imaging tools for tracking cell death immediately after treatment would not only be desirable for basic research but also of great benefit for evaluating therapy success. The caspases are a family of cysteine proteases that are critical for the AC710 execution of apoptosis. They are divided into two sub-families: the initiator caspases (caspases-2, -8, -9 and -10) which are initially activated by specific death stimuli from receptors or the mitochondria and the effector caspases (caspases-3, -6 and -7) that are brought on in response to initiator activation and overtake extensive substrate proteolysis leading finally to cellular destruction and death.4 Caspase-3 is a key mediator of the apoptotic process and the most proficient caspase, featuring an astonishing low inhibition of legumain in RAW cell lysates with the different inhibitors described in Table 1. (c) Chemical structure, fluorescence intensity and quenching efficiency of probes 17, 18 and 19. (d) Direct labeling of recombinant caspase-3 (upper panel) and legumain and cathepsin B in RAW AC710 cell lysate (lower panel) by indicated qABPs. Recombinant caspase-3 was incubated with increasing probe concentrations for one hour, the reaction was stopped and separated on a SDS PAGE and scanned for Cy5 fluorescence. Samples marked with + were pretreated with a caspase inhibitor (AB46 peptide) 30 min prior to the probe treatment. Legumain and cathepsin B from RAW cell lysates were labeled by the indicated qABPs similarly to caspase labeling. Samples marked with a, b or c were pretreated for 30 min with the inhibitors AB46 peptide, GB111-NH2?25 or 5 to selectively block caspase-3; cathepsin B or legumain, respectively. (e) Direct labeling of active caspase-3 in intact MM1s cells undergoing apoptosis. The indicated qABP showed covalent binding to active caspase-3, seen at 17 kDa. Samples marked with + represent the pretreatment with a caspase-3/legumain inhibitor (AB46 peptide) or cathepsin B inhibitor (GB111-NH2) which was added 1 h prior to the probe. Results Development of selective caspase-3 qABPs and their evaluation We set out to generate selective qABPs for caspase-3. We based our initial design on probes from the Bogyo group: AB46-Cy5, a non-quenched probe for caspase-3,18 AB50-Cy5?18 and LE28,27 and cathepsin quenched probes.25,26 AB46-Cy5 (Cy5-E8D-AOMK-DMBA, 8 stands for 2-amino butyric acid, see Table 1, bottom) was designed to be an ABP for caspase-3 but displayed cross-reactivity with legumain and cathepsin B. LE28 is usually a qABP based on AB50-Cy5 that targets both legumain and caspase-3 and contains a Cy5 fluorophore linked to a GluCProCAsp (P3CP2CP1) peptide scaffold and an acyloxymethyl ketone dimethylterephthalate propane linker attached to a quencher moiety (structures in Table 1, bottom). It is obvious that this cross reactivity to the two lysosomal cysteine proteases cathepsin B and legumain significantly lower the usage of caspase probes turning the development of more selective compounds highly attractable. Table 1 The compounds differ in their peptide sequence at the P2 position; R1 represents the corresponding side chain at this P2 position in the probe sequence E-P2-D. *R2 represents an acyl group or one of the two quenchers, QSY21 or BBQ (Blackberry quencher). **R3 indicates if the compound was fluorescently labeled or not. ***denotes the number of (CH2) models and thus the length of diaminolinker, % ACN denotes the percentage of acetonitrile at which the compound eluted from the analytical HPLC. All synthesized compounds were purified C-18 or C-4 preparative RP column after each synthetic step and characterized by LCMS. The final products were obtained in 3C32% yield after the final step of isolation and in over 95% purity (giving a single peak in the chromatogram at 215 as well as 254 nm) caspase-3 was comparable to compound 17C19, compound 22 was slightly less potent (Fig. 2a). However, almost no legumain and cathepsin.One day later, cells were treated with cisplatin (5 M) for 24 h. thus be used as an effective tool to study molecular mechanisms of programmed cell death in healthy and disease says. Introduction Apoptosis (programmed cell death) is the major process by which multi-cellular organisms eliminate excessive, damaged and potentially dangerous cells.1 Both normal and pathological processes such as embryonic development, malignancy, auto-immune disorders, ischemia and reperfusion or Parkinson’s and Alzheimer’s disease involve apoptotic cell death processes. Importantly, the induction of apoptosis in cancerous cells is the desired outcome of many cancer chemotherapeutic treatments and initiation or inhibition of apoptosis is usually a key element in numerous therapies.2,3 Therefore, the availability of imaging tools for tracking cell death immediately after treatment would not only be desirable for basic research but also of great benefit for evaluating therapy success. The caspases are a family of cysteine proteases that are critical for the execution of apoptosis. They are divided into two sub-families: the initiator caspases (caspases-2, -8, -9 and -10) which are initially activated by specific death stimuli from receptors or the mitochondria and the effector caspases (caspases-3, -6 and -7) that are brought on in response to initiator activation and overtake extensive substrate proteolysis leading finally to cellular destruction and death.4 Caspase-3 is a key mediator of the apoptotic process and the most proficient caspase, featuring an astonishing low inhibition of legumain in RAW cell lysates with the different inhibitors described in Table 1. (c) Chemical structure, fluorescence intensity and quenching efficiency of probes 17, 18 and 19. (d) Direct labeling of recombinant caspase-3 (upper panel) and legumain and cathepsin B in RAW cell lysate (lower panel) by indicated qABPs. Recombinant caspase-3 was incubated with increasing probe concentrations for one hour, the reaction was stopped and separated on a SDS PAGE and scanned for Cy5 fluorescence. Samples marked with + were pretreated with a caspase inhibitor (AB46 peptide) 30 min prior to the probe treatment. Legumain and cathepsin B from RAW cell lysates were labeled by the indicated qABPs similarly to caspase labeling. Samples marked with a, b or c were pretreated for 30 min with the inhibitors AB46 peptide, GB111-NH2?25 or 5 to selectively block caspase-3; cathepsin B or legumain, respectively. (e) Direct labeling of active caspase-3 Rabbit Polyclonal to RASD2 in intact MM1s cells undergoing apoptosis. The indicated qABP showed covalent binding to active caspase-3, seen at 17 kDa. Samples marked with + represent the pretreatment with a caspase-3/legumain inhibitor (AB46 peptide) or cathepsin B inhibitor (GB111-NH2) which was added 1 h prior to the probe. Results Development of selective caspase-3 qABPs and their evaluation We set out to generate selective qABPs for caspase-3. We based our initial design on probes from the Bogyo group: AB46-Cy5, a non-quenched probe for caspase-3,18 AB50-Cy5?18 and LE28,27 and cathepsin quenched AC710 probes.25,26 AB46-Cy5 (Cy5-E8D-AOMK-DMBA, 8 stands for 2-amino butyric acid, see Table 1, bottom) was designed to be an ABP for caspase-3 but displayed cross-reactivity with legumain and cathepsin B. LE28 is usually a qABP based on AB50-Cy5 that targets both legumain and caspase-3 and contains a Cy5 fluorophore linked to a GluCProCAsp (P3CP2CP1) peptide scaffold and an acyloxymethyl ketone dimethylterephthalate propane linker attached to a quencher moiety (structures in Table 1, bottom). It is obvious that this cross reactivity to the two lysosomal cysteine proteases cathepsin B and legumain significantly lower the usage of caspase probes turning the development of more selective compounds highly attractable. Table 1 The compounds differ in their peptide sequence at the P2 position; R1 represents the corresponding side chain at this P2 position in the probe sequence E-P2-D. *R2 represents an acyl group or one of the two quenchers, QSY21 or BBQ (Blackberry quencher). **R3 indicates if the compound was fluorescently labeled or not. ***denotes the number of (CH2) models and thus the length of diaminolinker, % ACN denotes the percentage of acetonitrile at which the compound eluted from the analytical HPLC. All synthesized compounds were purified C-18 or C-4 preparative RP column after each synthetic step and characterized by LCMS. The final products were obtained in 3C32% yield after the final step of isolation and in over 95% purity (giving a single peak in the chromatogram at 215 as well as 254 nm) caspase-3 was comparable to compound 17C19, compound 22 was slightly less potent (Fig. 2a). However, almost no.
Categories