Activation specific apoptotic caspases engineered

Orthogonal caspase activation by the SNIPer revealed synergy between caspase activation and pharmacological inhibition of the proteasome, and these data are consistent with a model in which the UPS potently restricts executioner caspase activity while the caspases disable the proteasome via subunit proteolysis during apoptosis.

Our new proteomics data reveals that caspase proteolysis of the proteasome subunits is more extensive than previously reported. Strikingly, caspase-6 can induce apoptosis only when activated in combination with proteasome inhibitors, and in this context the UPS appears to control the propagation of positive feedback from SNIPer-induced caspase activation to MOMP. This is the first example of reciprocal negative regulation between proteases that we are aware of.

Our data also provide an additional mechanism for bistability in apoptotic signaling. The caspase-6 zymogen is essentially converted to an apoptotic effector caspase by MG Recently, a targeted knockout of PSMC1 a 19S proteasome subunit in the mouse neuron results in the depletion the 26S proteasome and yielded a neurodegenerative-like phenotype including the presence of cleaved caspases Bedford et al.

Similarly, genetically encoded proteasome reporters are potently induced in the presence of pathogenic Huntington's Disease HD alleles Wang et al. Caspase-6 has already been implicated in the pathogenesis of the disease, but in the context of the present data, a second proteasome-caspase axis of mutual antagonism could exist in which HD patients could have impaired neuronal UPS function, stabilizing and potentially activating procaspase-6, which has been implicated in cleaving Htt Graham et al.

The proteasome inhibitor Bortezemib PS is approved as first-line therapy for treating multiple myeloma MM , a severe hematopoetic malignancy caused the clonal proliferation of plasma cells Raab et al.

Proteasome inhibitors induce apoptosis in these cell types Lee et al. Our model implies an additional mechanism: stabilization of the executioner caspases. As a single agent in MM, Bortezemib-mediated caspase stabilization may reduce the apoptotic threshold in these cells. Our data further suggest that combinations of proapoptotic agents to activate the caspases and proteasome inhibitors may synergize to induce apoptosis in additional cancer types.

Transient transfection of plasmids was performed at the or 6-well plates scale. Wells were coated with poly- d -lysine prior to plating target cells. Please see the Supplemental Experimental Procedures for the details of plasmid construction and Table S2 for all DNA oligonucleotide sequences used in this paper. Replicate wells in a well plate were mock-treated or with 10nM Rap. At defined endpoints, equal volume of either reagent was added to cells.

Cellular lysis proceeded at room temperature for 30 minutes, and luciferase activity was recorded in the SpectraMax M5 plate reader with an integration time of ms.

We are grateful to members of the Wells lab for useful discussions and to Emily Crawford for curation of the laboratory caspase substrate database. Kurt Thorn for assistance with live cell imaging. We also thank Drs. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript.

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Search articles by 'Daniel C Gray'. Gray DC 1 ,. Sami Mahrus Search articles by 'Sami Mahrus'. Mahrus S ,. Wells JA. Affiliations 1 author 1. A comment on this article appears in " SNIPer pulls the trigger. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases.

To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus TEV protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites.

Free full text. Author manuscript; available in PMC Jun PMID: Daniel C. Gray , 1, 3 Sami Mahrus , 1 and James A. James A. Author information Copyright and License information Disclaimer.

Copyright notice. The publisher's final edited version of this article is available at Cell. See other articles in PMC that cite the published article. Go to:. Supplemental Information. Open in a separate window. Figure 1. Engineering the SNIPer for Conditional Proteolysis A Generalized scheme for a ligand-inducible orthogonal protease based on the protein complementation system. Engineering Orthogonal Procaspase-3 and -7 Alleles for Selective Activation by the SNIPer The executioner caspases are translated as inactive zymogens that are cleaved by initiator caspases, by other executioner caspases in trans, or by auto-activation.

Figure 2. Selective Activation of Orthogonal Procaspase-3 and -7 Alleles in Stable Human Cell Lines The matrix of caspase-TevS alleles was stably engineered as single-copy integrants into HEK cells as described in the Supplementary Experimental Procedures in order to minimize the over-expression phenotypes associated with transient transfection.

Figure 3. Figure 4. Figure 5. Caspases Attack the 26S Proteasome, and Caspase Activation Synergizes with Proteasome Inhibition Recently gel-based proteomic data from several other laboratories have identified 10 proteins from the 26S proteasome, mostly in the 19S cap, that are cleaved during apoptosis Sun et al.

Figure 6. Orthogonal Activation of the Executioner Caspases Reveals Regulation by the UPS The SNIPer can be used to synchronize populations of cells expressing caspase alleles that mimic discrete states of caspase activation, and this approach has revealed the specific processing requirements for executioner caspase activation and maturation, which are difficult to deconvolute in cells Pop and Salvesen, Supplemental Information Click here to view.

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Dev Cell. Human caspases: activation, specificity, and regulation. Multiple myeloma. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase Negative autoregulation speeds the response times of transcription networks. Journal of Molecular Biology. Caspase activation, inhibition, and reactivation: a mechanistic view. Protein Sci. Caspase activation inhibits proteasome function during apoptosis.

Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol. Shotgun proteome analysis of protein cleavage in apoptotic cells.

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Targeted protein oxidation using a chromophore-modified rapamycin analog. Data Data behind the article This data has been text mined from the article, or deposited into data resources.

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