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Metacaspase Substrate Screening using Filter Aided Sample Preparation
Department of Plant Physiology, Umeå University, Umeå Plant Science Centre.
Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå Plant Science Centre.
Department of Plant Physiology, Umeå University, Umeå Plant Science Centre.
2011 (English)In: HUPO-2011: 10th World Congress of the Human Proteome Organization (HUPO): Geneva, Switzerland, September 3rd-7th, 2011: Proceedings-Posters, 2011Conference paper, Published paper (Refereed)
Abstract [en]

Background Metacaspases are proteases essential for programmed cell death (apoptosis) in plants, though most of their substrates remain unknown (1). The activity of metacaspases is a key issue in understanding wood (dead xylem cells) formation in trees. Here, we performed a substrate screening using a recombinant metacaspase that have an expression profile associated with the programmed cell death during wood formation in poplar. Methods Substrate preparation was initiated by total protein extraction from xylem tissue of plants down-regulated (RNA interference) in the expression of the metacaspase gene. Proper conditions intended for screening of the enzyme substrates were achieved by combinations of enzyme and substrate ultrafiltration. Utilizing the low pH activation of the enzyme allowed prompt activation and minute monitoring of newly formed peptides. Peptides were analysed by LC-MSMS using a nano-LC system coupled to a Synapt G2 mass spectrometer. Additional extractions including wild-type plants were performed to support detected substrate processing. Results The results revealed an enzyme active at low temperature (< 7 °C) with properties fitting criteria for cold-adapted enzymes. High specific activity was detected at low temperature and degradation products from the enzyme were formed after 30 min incubation at room temperature. Conclusions Our preliminary data suggest a substrate that has global responses that would clarify the metacaspase involvement in programmed cell death and wood formation. 1.Sundstrom, J. F., Vaculova, A., Smertenko, A. P., Savenkov, E. I., Golovko, A., Minina, E., Tiwari, B. S., Rodriguez-Nieto, S., Zamyatnin, A. A., Valineva, T., Saarikettu, J., Frilander, M. J., Suarez, M. F., Zavialov, A., Stahl, U., Hussey, P. J., Silvennoinen, O., Sundberg, E., Zhivotovsky, B., and Bozhkov, P. V. (2009) Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome. Nat. Cell Biol. 11, 1347-U198.

Place, publisher, year, edition, pages
2011.
Identifiers
URN: urn:nbn:se:ltu:diva-32268Local ID: 6b54379d-ffbd-490f-bef7-139aadb9afa1OAI: oai:DiVA.org:ltu-32268DiVA, id: diva2:1005502
Note
Upprättat; 2011; 20130415 (robnil)Available from: 2016-09-30 Created: 2016-09-30 Last updated: 2018-05-28Bibliographically approved

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