1. Volume 8, Issue 3, Pages 613-621 (September 2001)
A Serine Protease, HtrA2, Is Released from the Mitochondria and Interacts with XIAP, Inducing Cell Death 
Yasuyuki Suzuki, Yuzuru Imai, Hiroshi Nakayama, Kazuko Takahashi, Koji Takio, Ryosuke Takahashi 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1 Detection of XIAP Binding Protein and Identification of HtrA2
(A) Overexpressed FLAG-XIAP lacking the RING finger motif (XIAPΔRING) in 293T cells was affinity purified using anti-FLAG (M2) agarose (Sigma). Eluted fractions were subjected to SDS-PAGE and then stained with Coomassie blue. The arrowhead indicates purified FLAG-XIAPΔRING. Coeluted proteins are represented by the letters A and B (see text).
(B) RP-HPLC of Lys-C-digests of the 36 kDa XIAP binding protein (protein band A in Figure 1A). Amino acid sequence analysis by Edman degradation of fraction 1 revealed that this fraction contains the N-terminal fragment of mature HtrA2 (the underlined sequence in Figure 1C). Based on this information, fractions 2, 3, 4, 5, and were shown to correspond to amino acids 325–347, 238–324, 349–395, 157–237, and 396–458 of full-length HtrA2 protein, respectively.
(C) Amino acid sequence of the full-length 13B form of human HtrA2. The sequence of fraction 1 in Figure 1B is underlined. The arrowhead indicates the site at which cleavage occurs, thereby generating mature HtrA2
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Interaction between HtrA2 and IAP Family Proteins
(A) Endogenous XIAP associates with endogenous HtrA2. Whole-cell lysates (WCL) from HEK293 cells (1 mg protein) were incubated with 1 μg of control mAb and anti-XIAP mAb conjugated with 5 μl of protein G-Sepharose at 4°C for 4 hr. Immunoprecipitates (IP) were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting (IB) using anti-HtrA2 and anti-XIAP rabbit polyclonal Ab. The bands for the heavy chain and the light chain of IgG are indicated as IgG HC and IgG LC, respectively.
(B) Mature HtrA2 but not its precursor associates with IAP family proteins. N-terminal FLAG-tagged XIAP, c-IAP1, c-IAP2, Myc-tagged XIAP, and survivin cDNA were transfected with C-terminal HA-tagged full-length HtrA2 cDNA into HEK293 cells. Whole-cell lysates (WCL) and immunoprecipitates with anti-FLAG mAb (IP: FLAG) or anti-Myc mAb (IP: Myc) were analyzed by immunoblotting (IB) using anti-FLAG, anti-Myc, or anti-HA mAb
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Mature HtrA2 but Not Its N-Terminal Missense Mutant Binds to and Inhibits XIAP in a Similar Manner to Smac
(A) Schematic representation of full-length HtrA2 (HtrA2-FL) and mature HtrA2. The transmembrane (TM) segment, the trypsin-like catalytic domain, and the PDZ domain are located among amino acids 105–121, 182–330, and 390–445 of HtrA2-FL, respectively. The serine residue of the active site is labeled S306. RecHtrA2(AVPS) and recHtrA2(MVPS) designate the bacterially produced recombinant proteins of wild-type and N-terminal mutant HtrA2, respectively. Although the N-terminal sequence of RecHtrA2(AVPS) was originally constructed as MAVPS, the initial methionine is cleaved off in bacteria, generating wild-type mature HtrA2 protein.
(B) RecHtrA2(AVPS) but not recHtrA2(MVPS) binds to the BIR2 and BIR3 regions of XIAP. Recombinant wild-type and mutant HtrA2 protein was pulled down with a series of GST-fused XIAP deletion mutants, after which immunoblot analysis was performed using anti-penta His or anti-GST Abs. The arrowhead indicates C-terminal His6-tagged recombinant mature HtrA2.
(C) Schematic representation of the processing of procaspase-3 and the points at which XIAP fragments inhibit this processing. The BIR3 + RING (BIR3+R) fragment inhibits the processing of procaspase-3 (p32) by caspase-9 to generate p20 and p12 fragments, while the BIR2 fragment inhibits the processing of p20 by caspase-3 to yield p19 or p17 fragments.
(D) RecHtrA2(AVPS) but not recHtrA2(MVPS) inhibits both caspase-3 inhibition by GST-BIR2 (BIR2) and caspase-9 inhibition by GST-BIR3 + RING (BIR3+R). Cytosolic extracts prepared from HEK293 cells were incubated with cytochrome c/dATP to induce caspase-3 activation in vitro in the presence or absence of the indicated proteins. Following this, the extracts were subjected to SDS-PAGE followed by immunoblot with anti-caspase-3 polyclonal Ab, as described elsewhere (Deveraux et al., 1999; Takahashi et al., 1998). The open arrowhead indicates contamination with a small amount of protein from E. coli which crossreacts with anti-caspase-3 Ab
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 XIAP Does Not Inhibit the Serine Protease Activity of HtrA2
Coomassie blue-stained polyacrylamide gel of His6-tagged recombinant HtrA2 after 1 hr and 2 hr of incubation. β-casein (6 μM) as a generic substrate was incubated with 50 nM recombinant HtrA2 serine protease in the presence of the indicated proteins. Even when incubated with 2 μM GST-XIAP, the amount of cleaved β-casein was almost identical to that obtained from incubation without XIAP
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
HtrA2 Is Released from Mitochondria to the Cytosol in Response to UV Irradiation
(A) Subcellular fractionation of HeLa cells. HtrA2 and cytochrome c were observed to colocalize within the fraction containing mitochondria. Protein disulfide isomerase (PDI) and XIAP represented the endoplasmic reticulum resident protein and the cytosolic protein, respectively (Duckett et al., 1996; Ferrari and Soling, 1999). Although PDI was detected in both the heavy membrane fraction and cytosolic extract, presumably due to leakage, its presence in the light membrane fraction containing the endoplasmic reticulum was evident. HM, heavy membrane fraction; LM, light membrane fraction; CE, cytosolic extract.
(B) Immunostaining of HtrA2 and cytochrome c. Before and 4 hr after UV (100 mJ/cm2) irradiation, the cells were fixed and immunostained with anti-HtrA2 and anti-cytochrome c mAb.
(C) Cytosolic translocation of HtrA2 and cytochrome c after UV irradiation. Cytosolic extracts were prepared from UV (100 mJ/cm2) irradiated cells at the time indicated and immunoblotted with anti-HtrA2 polyclonal Ab, anti-cytochrome c mAb, or anti-actin mAb
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 HtrA2 Enhances DEVDase Activity Induced by UV Irradiation
HeLa cells were cotransfected with 0.8 μg of pcDNA3 (Mock), pcDNA3-Smac-Myc (Smac), or pcDNA3-HtrA2-Myc (HtrA2) together with 0.2 μg of pEGFP plasmid, incubated for 24 hr, then stimulated by UV (100mJ/cm2) irradiation. After 2 or 4 hr, cell lysates were prepared, and DEVDase activity within 30 μg of lysate from each preparation was measured. The error bars represent the SE calculated from triplicate samples
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7
Extramitochondrially Overexpressed HtrA2 Induces Caspase-Independent Atypical Cell Death
(A) The expression of mature HtrA2 induces atypical cell death. HEK293 cells were cotransfected with 1 μg of pcDNA3 (Mock) or pcDNA3-mature HtrA2-Myc (mature HtrA2) together with 0.2 μg of pEGFP plasmid. After 30 hr, GFP-positive cells were visualized by confocal laser microscopy.
(B) Protease activity of mature HtrA2 is required for atypical cell death. HEK293 cells were cotransfected with pcDNA3 (Mock), pcDNA3 encoding mature HtrA2-Myc (mature HtrA2), active site Ser to Ala mutant of mature HtrA2-Myc [mature HtrA2(S/A)], full-length HtrA2-Myc (FL-HtrA2), or full-length Smac-Myc (FL-Smac) along with pEGFP plasmid as in (A). After 24 hr, GFP-positive cells with round or shrunken shapes were counted. The error bars represent the SE calculated from triplicate samples.
(C) DEVDase activity is not significantly induced by mature HtrA2. HEK293 cells were cotransfected with the indicated constructs along with pEGFP plasmid as in (A). After 24 hr, cell lysates were prepared and DEVDase activity in 100 μg of each lysate sample was measured. The error bars represent the SE calculated from triplicate samples.
(D) Caspase inhibitors cannot block the effect of mature HtrA2. HEK293 cells were cotransfected with the indicated constructs along with pEGFP plasmid as in (A). During transfection and following incubation, the broad-spectrum caspase inhibitor zVAD-fmk (100 μM) was added as indicated. The error bars represent the SE calculated from triplicate samples
Molecular Cell 2001 8, DOI: ( /S (01) )