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CED-9

CED-9. A Caenorhabditis Elegans Anti-apoptotic BCL-2 Homologue Presentation by Peter Audano. Apoptosis. Apoptosis is programmed cell death. Cells are quickly engulfed by their neighbors. Why? Damaged or aberrant cells. Development. Without apoptosis…

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CED-9

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  1. CED-9 A Caenorhabditis Elegans Anti-apoptotic BCL-2 Homologue Presentation by Peter Audano

  2. Apoptosis • Apoptosis is programmed cell death. • Cells are quickly engulfed by their neighbors. • Why? • Damaged or aberrant cells. • Development. • Without apoptosis… • Potentially dangerous dysfunctional cells. • Post-developmental clutter. • Cancer.

  3. CED-9 Overview • CED-9 gene codes for a protein. • Apoptosis inhibitor. • Loss of CED-9 in C. elegans results in excessive cell death and increased CED-9 activity results in little or no cell death. • Transmembrane, mitochondria bound (1). • Interacts with other proteins that promote or inhibit cell death.

  4. CED-9 Expression • Expressed in gonads. • Most prevalent 250-500 minutes after fertilization (1). (3)

  5. 131 cells die during hermaphrodite development (1). Almost all cell death occurs during embryogenesis (1). Why study apoptosis C. elegans? Homologues in humans. Key role in deciphering apoptotic machinery in mamals. Apoptosis in C. elegans

  6. Apoptosis in C. elegans (2)

  7. Apoptosis in C. elegans • CED-3: A protease that actively degrades other proteins. • Active CED-3 triggers apoptosis. • CED-4: Complexes with a polypeptide zymogen to assist CED-3 formation (6). • P-loop mediated. • Processes inactive proCED-3 to active CED-3. • CED-9: Complexes with CED-4 and prevents it from creating CED-3 (6). • Contacts CED-4 P-loop and inhibits its interaction with the CED-3 prodomain (6). • Binds CED-4 to mitochondria membrane (1). • EGL-1: Complexes with CED-9 and inhibits its ability to complex with CED-4.

  8. CED-4 Inhibition • CED-4 is P-loop mediated. • A P-loop is an ATP/GTP binding site. It is “Glycine-rich loop preceded by a β-sheet and followed by an α-helix” (7). • If CED-4 is inactivated by removing a critical lysine or by insertion inactivation, it cannot mediate CED-3 processing. This provides evidence that interfering with the P-loop inhibits CED-4. • CED-9 inactivates and binds CED-4 to mitochondria where it cannot process proCED-3.

  9. CED-9 Inhibition • EGL-1 binds to CED-9 and disrupts CED-9/CED-4 interaction (1). • This unbinds CED-4 and frees it to process pro-CED3 into CED-3. • Cells destined for apoptosis during development express EGL-1.

  10. Apoptosis in C. elegans (2)

  11. CED-9 Sequence • 843 coding nucleotides. • 2782 nucleotides in transcript. • Exons, Introns, and 3` and 5` UTR. • 5 exons, 4 introns. • One intron appears outside of coding region. • Located on Chromosome III • 10334642-10337423

  12. CED-9 Sequence - Wormbase (1)

  13. CED-9 Sequence - EMBL (9) (9) (9)

  14. CED-9 BLAST - Nucleotide • A small region is highly conserved among many C. elegans genes. • Conserved region is an intron in CED-9. • Doesn’t appear to be a repeated sequence.

  15. CED-9 BLAST - Nucleotide • All results were C. elegans. Highest non-CED-9 sequences had E values of 7e-57 and higher.

  16. CED-9 Alleles • 10 alleles (1). • n1653: Abnormal sexual development. • n1950: G/A substitution. • n1950n2077 • n1950n2161: Increased cellular death. • n2161 • n2812 • niDf71(III): bp deletion. • ok909: 1071 bp deletion + GAAA insertion. • pas48890: 2 bp deltion. • tm1081: 774 bp deletion, TTTT insertion. Lethal or sterile.

  17. CED-9 Protein • Cell surface, trans-membrane protein. • Class: Mainly α-helices. • Structure: Orthogonal bundle. • Structure verified by crystallization. CED-9 CED-9/EGL-1 Complex CED-9 inactive CED-4/CED-9 Complex CED-4 inactive (4) (4) (4)

  18. CED-9 Protein Structure • Consists of α-helices (red spirals) and turns (yellow arches). (4)

  19. CED-9 Protein BLAST (8)

  20. CED-9 Protein BLAST • Top results are CED-9. • BCL-2 family • E = 1e-22 • BCL2-like proteins • Rattus norvegicus (Norway rat) • E = 1e-06 • Mus musculus (mouse) • E = 2e-06 • KIAA0271 • Homo sapiens apoptosis regulator • E = 3e-6 (8)

  21. BCL-2 Family • BCL-2: A family of proteins found in humans and are part of the apoptosis pathways. • CED-9 and EGL-1 homologues in humans. • 16+ members. • Some prevent cell death, and some promote it. • Proto-oncogene. Dysfunction prevents normal cell death and can lead to cancer. • Can replace CED-9 in C. elegans. • Functionality is highly conserved.

  22. Resource Summary • Wormbase • The most complete resource for this gene. • DDBJ and SwissPROT contained much of the Wormbase findings. • BLAST • Nucleotide search: Not much interesting information. • Protein search: Revealed many CED-9 homologues, especially in the BCL-2 family. • PDB • Most complete polypeptide information, including structure and function. • Science, Nature, Cell, Wormbook, and NCBI • Most complete published resources available.

  23. CED-9 Discovery • I was not able to find who or how the gene was discovered. • Hentgartner and Horvitz did a great deal of work on C. elegans apoptosis, but I could not access some of their papers. I can only assume they were involved in the discovery of CED-9. • CED-9 is verified with cDNA evidence.

  24. Hentgartner, M (n.d.). Roads to Ruin: Apoptotic Pathways in the Nematode Caenorhabditis Elegans. Retrieved 4/26/2008 from http://www.cloetta-stiftung.ch/Hengartner_Ref.pdf Vaskivuo, T. (2002). Regulation of Apoptosis in the Female Reproductive System. Retrieved 4/26/2008 from http://herkules.oulu.fi/isbn9514266676/html/i267425.html Wormbase (n.d.). Gene Summary for ced-9. Retrieved 4/26/2008 from http://www.wormbase.org/db/gene/gene?name=WBGene00000423;class=Gene RCSB Protein Data Bank (n.d.). 1ty4. Retrieved 4/26/2008 from http://pdb.rcsb.org/pdb/explore.do?structureId=1TY4, http://pdb.rcsb.org/pdb/explore.do?structureId=2A5Y, and http://pdb.rcsb.org/pdb/explore.do?structureId=1OHU Horvitz, B., Hengartner, M. (n.d.). No Title. Retrieved 4/26/2008 from http://www.dnalc.org/horvitz.swf Chaudhary, D., O’Rourke, K., et. al. (1998). The Death Inhibitory Molecules CED-9 and CED-4L Use a Common Mechanism to Inhibit the CED-3 Death Protease. Retrieved 4/26/2008 from http://www.jbc.org/cgi/content/full/273/28/17708 Sources

  25. Sources • P-loop. Wikipedia (n.d.). Retrieved 04/26/2008 from http://en.wikipedia.org/wiki/P-loop • NCBI BLAST (n.d.). Retrieved 04/26/2008 from http://www.ncbi.nlm.nih.gov/blast/Blast.cgi • EMBL. Ensembl Gene Report for T07C4.8 (n.d.). Retrieved 4/27/2008 from http://www.ensembl.org/Caenorhabditis_elegans/geneview?gene=T07C4.8 • DDBJ (n.d.). ARSA Search. Retrieved 4/17/2008 from http://www.ddbj.nig.ac.jp/

  26. Questions? Austin Powers Says… Questions are groovy baby, yea.

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