Progress Report Apoptosis inducing factor l l Introduction

Progress Report Apoptosis inducing factor l l Introduction Experiment

Introduction Apoptosis inducing factor (AIF) E. Daugas et al. /FEBS Letters 476 (2000) 118 -123 The detailed position of the m. AIF gene was mapped to chromosome X region A 6 The detailed position of the h. AIF gene was mapped to chromosome Xq 25– 26

Characteristic domain of h. AIF Primary structure of h. AIF TRENDS in Pharmacological Sciences Vol. 25 No. 5 May 2004 • • • 1 -100: N-terminal mitochondrial localization sequence, MLS 101 -127: spacer 128 -613: C-terminal oxidoreductase domain (including a nuclear localization sequence, NLS) • MW of pro. AIF is 67 k. D, MW of mature AIF is 57 k. D

Crystal structure • FAD-binding domain 128 -262 and 401 -480 • NADH-binding domain 263 -400 • C-terminal domain 481 -608 Nature Structural Biology 9, 680 - 684 (2002)

Functions • Oxidoreductase: Mitochondrial flavoprotein with NADH oxidase activity • Apoptosis: It induces chromatin condensation and high-molecular-weight (50 kbp) DNA fragmentation ※ AIF binds to DNA and RNA C. Candé et al. / Biochimie 84 (2002) 215– 222

DNA binding assay in vitro in vivo protein/DNA fragment molar ratios 10 30 Oncogene (2005), 1– 12 50

RNA binding assay in vitro in vivo Oncogene (2005), 1– 12

Apoptosis pathway l Caspase dependent l Caspase independent

Caspase dependent cell death TRENDS in Pharmacological Sciences Vol. 25 No. 5 May 2004

Caspase independent cell death TRENDS in Pharmacological Sciences Vol. 25 No. 5 May 2004

The roles of AIF in cell death AIF is co-localization with DNA during apoptosis Normal apoptosis Nature structure biology．Volume 9 number 9．September 2002

DNA binding of AIF is required for apoptosis • DNA binding of mutant AIF Nature structure biology．Volume 9 number 9．September 2002

AIF associated with Endo. G Nickel-affinity pull-down assay • Endonuclease G (Endo. G) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. Endo. G will digest either DNA or RNA. Apoptosis 2005; 10: 821– 830

WAH-1 cooperates with CPS-6 to promote DNA degradation • WAH-1 is homolog of AIF • CPS-6 is homolog of endo. G • oc sc 22 NOVEMBER 2002 VOL 298 SCIENCE

• What do we want to know? How does AIF promote activity of endo. G? • What can we do? Co-crystallization with endo. G Journal of Cellular Biochemistry 94: 1078– 1087 (2005)

Experiment 1. He. La cell → total RNA → RT PCR → AIF 2. TA cloning 3. Construct: AIF/p. QE 30 Expression strain: B 834, M 15, BL 21 p. Lys, BL 21 RIPL, JM 109 4. Construct: AIF 121/p. ET 28 a Expression strain: BL 21 p. Lys, BL 21 RIPL

AIF/p. QE 30 37℃, 3 hrs, 1 m. M IPTG induction B IPTG - total M P R - J - B + total M P R + + + J +

30℃, 8 hrs, 1 m. M IPTG induction B IPTG - total M P R - J - B + total M P R + + + J +

25℃, 14 hrs, 1 m. M IPTG induction B IPTG - total M P R - J - B + M + total P R + + J +

20℃, 20 hrs, 1 m. M IPTG induction B IPTG - total M P - R J - - B + M + total P R + + J +

AIF 121/p. ET 28 a/BL 21 p. Lys 37℃, 3 hrs, 1 m. M IPTG induction IPTG total + medium + pellet +

30℃, 8 hrs, 1 m. M IPTG induction total IPTG - + medium + pellet +

25℃, 14 hrs, 1 m. M IPTG induction total IPTG - + medium + pellet +

20℃, 20 hrs, 1 m. M IPTG induction IPTG medium + pellet - +

AIF 121/p. ET 28 a/BL 21 RIPL 37℃, 3 hrs, 1 m. M IPTG induction IPTG total + pellet + total medium + IPTG 70 70 55 55 - + pellet + medium +

25℃, 12 hrs, 1 m. M IPTG induction total IPTG - + medium + pellet - +

Future work l l Purification of h. AIF Co-crystallization with endo. G