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PINK1 cleavage at position A103 by the mitochondrial protease PARL

PINK1 cleavage at position A103 by the mitochondrial protease PARL. Emma Deas , Helene Plun-Favreau , Sonia Gandhi, Howard Desmond, Svend Kjae , Samantha H.Y. Loh , Alan E.M. Renton, Robert J. Harvey, Alexander J. Whitworth, L. Miguel Martins, Andrey Y. Abramov and Nicholas W. Wood

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PINK1 cleavage at position A103 by the mitochondrial protease PARL

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  1. PINK1 cleavage at position A103 by the mitochondrial protease PARL Emma Deas, Helene Plun-Favreau, Sonia Gandhi, Howard Desmond, SvendKjae, Samantha H.Y. Loh, Alan E.M. Renton, Robert J. Harvey, Alexander J. Whitworth, L. Miguel Martins, Andrey Y. Abramov and Nicholas W. Wood Human Molecular Genetics, 2011, Vol. 20 No. 5, Page 867- 879 Sharif Abu Hayat

  2. PINK 1

  3. Objectives • Determination of the cleavage site of PINK1 • Mutational analysis of cleavage site residues • Observation of PD associated mutations • Cellular consequences of impaired PINK1 • Identification of the cleavage protease

  4. PINK1 Cleavage site determination

  5. PINK1 Cleavage site determination

  6. WB analysis • Sequencing result • conservation in mammals

  7. Mutational analysis of cleavage site

  8. Observation of PD associated mutations

  9. Impaired PINK1: Cellular consequences • TMRM fluorescent intensity measurement • Mitochondrial membrane potential, ΔѰm

  10. Impaired PINK1: Cellular consequences Generation of harmful ROS Cytosolic hydroethidium (HEt) fluorescence MitoSOX fluorescence

  11. Impaired PINK1: Cellular consequences Stimulation of ROS production using rotenone

  12. Impaired PINK1: Cellular consequences Normal mitochondrial network Mitochondrial TMRM Cytosolic GFP

  13. Impaired PINK1: Cellular consequences Disrupted mitochondrial network Cytosolic GFP TMRM

  14. Impaired PINK1: Cellular consequences Loss of mitochondrial mass Co-localization of the mitochondrial (DsRed-Mito) signal with the cytosolic (GFP)

  15. Impaired PINK1: Cellular consequences No variation in basal and CCCP-induced levels of LC3 I-II cleavage

  16. PARL is the protease responsible for the cleavage of PINK1: 1. High temperature requirement protein A2 (HtrA2) 2. Presenilin-associated rhomboid-like protein (PARL)

  17. PARL is the protease responsible for the cleavage of PINK1:

  18. PARL is the protease responsible for the cleavage of PINK1:

  19. Conclusion • Disruption of distribution of the mitochondrial network • Reduction in mitochondrial mass inside the cell independent of mitophagy activation • Lowering of Mitochondrial membrane potential, ΔѰm • Increase in generation of harmful ROS • An increased ratio of FL- to ΔN-PINK1, expresses intermediate mitochondrial phenotype

  20. Future Research: • Cleavage recognition site for PARL • ΔN2-PINK1 • Alternative route to LC3 I-II proteasome • Modulated expression of ΔN-PINK1

  21. Thank You!

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