Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3.
Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D(3)A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D(3)A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D(3)A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both (16)O/(18)O- and (14)N/(15)N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D(3)A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.
Molecular Medicine Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC.
Y-J
YJ
Hsieh
K-Y
KY
Chien
S-Y
SY
Lin
S
S
Sabu
R-M
RM
Hsu
L-M
LM
Chi
P-C
PC
Lyu
J-S
JS
Yu
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D017323:Dihematoporphyrin Ether / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D019169:Jurkat Cells; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes / Q000737:chemistry; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
D009587:Nitrogen Isotopes; D010103:Oxygen Isotopes; D017319:Photosensitizing Agents; D011994:Recombinant Proteins; D017323:Dihematoporphyrin Ether; D008715:Methionine; D053148:Caspase 3