Potential cell death cleavage sites highlighted in bovine PARP amino acid sequence.

A. Amino acid sequence of bovine PARP

ORIGIN

10 20 30 40 50 60

MAESSDKLYRVEYAKSGRAS CKKCKESIPKDSIRMAFMVE SPMFDGKIPH WYHLSCFWKV

70 80 90 100 110 120

GFSIWHPDVE VEGFSELRWD DQQTIKKMAE TGGRTDVSGK GQDGVGSKTE KTLIDFGAGY

130 140 150 160 170 180

AKSNRSTCKS CMEKIDKGQV RLSKKVVYPD KPQLGMVDCW YHPKCFVQKR EELGFRPEFS

190 200 210 220 230 240

ATHLMGFSVL TAEDQETLKK QLPAIKGERK RKGDEVDGIDEVTKKKSKKE KDKEIKLEKA

250 260 270 280 290 300

LKAQNDLIWN VKDELKKACS TNDLKELLIF NKQEVPSGES AILDRVADGMVFGALLPCEE

↓ 310 320 330 340 350 360

CSGQLVFKGDAYYCTGDVTA WTKCMVKTQT PNRKEWVTPK EFREISYFKKLKIKKQDRIF

370 380 390 400 410 420

PPESSTPVGA AAPPSAASAP AAVHSGPPDKPLSNMKILTL GKLSQNKDEV KATIEKLGGK

430 440 450 460 470 480

LTGTANKASL CISTKKEVDK LNKKMEEVKE ANIRVVSEDF LQDISASTKS LQELLSTHLL

490 500 510 520 530 540

SPWGAEVKVEPVEAVGPKGKSGAAPSKKSKGPVKEEGTNK SEKRMKLTLKGGAAVDPDSG

550 560 570 580 590 600

LEHNAHVLEK GGKVFSATLG LVDIVKGTNS YYKLQLLEDDKESRYWIFRSWGRVGTVIGS

610 620 630 640 650 660

NKLEQMPSKE DAIEHFMKLYEEKTGNAWHS KNFTKHPKKF YPLEIDYGQDEEAVKKLTVN

670 680 690 700 710 720

PGTKSKLPKPVQNLIKMIFD VESMKKAMVEYEIDLQKMPLGKLSKRQIQA AYSILSEVQQ

730 740 750 760 770 780

ALSQGSSDSH ILDLSNRFYT LIPHDFGMKKPPLLNNANSV QAKVEMLDNL LDIEVAYSLL

790 800 810 820 830 840

RGGSDDSSKD PIDVNYEKLK TDIKVVDKDSEEAEIIRKYV KNTHATTHNA YDLEVVDIFK

850 860 870 880 890 900

IEREGESQRY KPFKQLHNRRLLWHGSRTTN FAGILSQGLR IAPPEAPVTG YMFGKGIYFA

910 920 930 940 950 960

DMVSKSANYC HTSQGDPIGL ILLGEAALGN MYELKHARHI SKLPKGKHSV KGLGKTTPDP

970 980 990 1000 1010

SASITVDGVEVPLGTGISSG VNDTCLLYNEYIVYDIAQVH LKYLLKLKFN FKTSLW//

END.*Cleavage sites based on synthetic substrates and literature.

B.

Legend of highlighted potential cleavage sites
FR / cathepsin B & L
DEVD / caspase 3
VAD / partial caspase 2
S_QLV__G / putative N-terminus of our fragment
IXXD, DXD / granzyme B
LY / calpain class I and II
H384, L480, G502, T658 / calpain*
RR, T528, T558 / cathepsin B [25]
L527, K555 / cathepsin G [24]
LEXD / caspases -8, -9, 10
YEXD / caspases -1, -4, -5, 14

* Buki, K. G., Bauer, P. I., and Kun, E. (1997) Biochim, Biophys. Acta 1338:100-106.

* Tompa, P., Buzder-Lantos, P., Tantos, A., et al (2004) J. Biol. Chem. 279: 20775-20785.

Figure S1. Potential cell death cleavage sites highlighted in bovine PARP amino acid sequence.

Our ~50 kDa PARP fragment was sent to the W.M. Keck Biotechnology Resource Lab at Yale University, and the sequence was read by Nancy Williams. Results were compared to the amino acid sequence for bovine PARP obtained from the National Center for Biotechnology Information (NCBI) Sequence Viewer: =protein&val=130779. Potential cleavage sites for various enzymes are highlighted throughout the PARP sequence (A). Sites correspond to either commercially available synthetic substrates or cleavage sites mentioned in the literature (see legend, B). Amino acids matching the results of the first 5 cycles of Edman (N-terminal) sequencing are highlighted beginning at a.a. 302 (S*GLV**G). Hence, the putative N-terminus of our fragment does not correspond to any of the possible sites we have located within the sequence. The suggestion is that more than one protease, including perhaps an exopeptidase, is active during the peak period of degeneration.