1. The Mitochondrial Rhomboid Protease PARL Is Regulated by PDK2 to Integrate Mitochondrial Quality Control and Metabolism 
Guang Shi, G. Angus McQuibban 
Cell Reports 
Volume 18, Issue 6, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1458-1472DOI: (10.1016/j.celrep.2017.01.029)
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3. Figure 1 PARL β Cleavage Is Induced by Mitochondrial Damage
(A) β cleavage after 24-hr treatments of DMSO, 20 μM Oligomycin A (OL), 25 μM Rotenone (RT), 20 μM CCCP, and 1 μM Thapsigargin (TA) in HEK293 cells overexpressing PARL-FLAG. β cleavage produces a cleaved form of PARL (PACT ∼28 kDa). β cleavage level in each condition was calculated by comparing the signal intensity of PACT to total PARL signal. Mitochondrial protein ATP5A was used as loading control.
(B) Quantification of the β cleavage level in each condition in (A). Relative to DMSO treatment, a significant increase in the level of β cleavage was observed in all treatments except TA.
(C) Measurements of mitochondrial ATP level in each treatment in (A). All treatments, except TA, significantly reduced mitochondrial ATP level to ∼60% of DMSO treatment.
Results in (B) and (C) were normalized to DMSO control. ∗p < 0.01, ∗∗p < 0.02, ∗∗∗p <
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4. Figure 2 β Cleavage Generates PACT that Is Catalytically Less Active
(A) PINK1 was transiently transfected into PARL+/+ and –/– MEFs (lanes 1 and 2, respectively). PINK1 and wild-type (WT) PARL, S277G PARL catalytic mutant, S77N PARL mutant that impairs β cleavage, and PACT, a product from β cleavage, were co-transfected into PARL–/– MEFs (lanes 3–6, respectively). Cells were treated with 10 μM MG132 16 hr prior to lysis to stabilize PINK1. Expression of PARL constructs was confirmed by WB (middle panel). Full-length PINK1 (∼68 kDa, FL-PINK1) and processed PINK1 (∼58 kDa, Δ-PINK1) were observed in the presence of PARL. Accumulation of full-length PINK1 as well as PARL independent PINK1-processing products (∗∼54 and ∼50 kDa) were detected in the absence of PARL. Expression of WT PARL in PARL–/– MEF eliminated the accumulation of full-length PINK1. However, expression of the catalytically inactive PARL S277G mutant did not. Expression of S77N PARL and PACT, similar to WT PARL, restore the PINK1-processing pattern.
(B) PINK1 was co-transfected with increasing amounts (0.25, 0.5, and 1 μg) of WT PARL, S77N PARL, and PACT into PARL–/– MEFs, respectively. Cells were treated with 10 μM MG132 16 hr prior to lysis. Expression of PARL constructs was confirmed by WB (middle panel). Mitochondrial protein ATP5A was used as a loading control to ensure equal loading (bottom panel). Compared to untransfected cells (lane 1), expression of WT and S77N PARL, as low as 0.25 μg DNA (lanes 2 and 6), effectively leads to PINK1 cleavage to generates Δ–PINK1; at the same concentration, PINK1 processing was not observed in cells transfected with PACT (lane 10).
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5. Figure 3 Depletion of Mitochondrial ATP Triggers β Cleavage
(A) β cleavage in HEK293 cells that overexpress PARL-FLAG after 24-hr treatment of increasing concentrations of OL. The mitochondrial protein ATP5A was used as a loading control.
(B) Quantification of the β cleavage level in (A). Compared to the control, a significant increase in β cleavage was observed in cells treated with 15 and 20 μM OL.
(C) Measurements of mitochondrial and cellular ATP in each treatment in (A). No significant changes in cellular ATP levels were observed among samples, whereas a significant reduction in mitochondrial ATP was observed between the control and the cells treated with 12.5 μM and a higher concentration of OL.
(D) β cleavage levels in SH-SY5Y cells after 24-hr treatment of increasing concentrations of OL. Immunoprecipitation (IP) was performed on cell lysates to concentrate PARL signal. Relative to the control, where β cleavage is undetectable (lane 1), β cleavage is induced with OL treatment as low as 2.5 μM.
(E) Measurements of mitochondrial ATP in each treatment in (D). A significant reduction in mitochondrial ATP was observed in samples that treated with as low as 2.5 μM of OL.
Results in (B), (C), and (E) were normalized to 0 μM OL treatment. ∗p < 0.01, ∗∗p < 0.02, ∗∗∗p < See also Figure S1.
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6. Figure 4
Inhibition of Pyruvate Dehydrogenase Kinase Induces β Cleavage
(A) β cleavage in HEK293 cells that overexpress PARL after 24-hr treatment of increasing concentrations of DCA and MOX. The mitochondrial protein ATP5A was used as loading control.
(B) Quantification of the β cleavage level in (A). Compared to the control (0 μM inhibitor treatment), β cleavage in cells that treated with 0.1 mM DCA was significantly increased (lanes 1 and 3). No significant change was observed between cells that were treated with and without MOX treatment.
(C) β cleavage in SH-SY5Y cells after 24-hr treatment of increasing concentrations of DCA and MOX. Immunoprecipitation (IP) was performed on cell lysates to concentrate the PARL signal.
(D) Quantification of the β cleavage level in (C). DCA treatment induces β cleavage with concentration as low as 0.05 mM, while MOX treatment did not alter the β cleavage level at any concentrations.
Results in (B) and (D) were normalized to the control. ∗∗∗p < also Figure S2.
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7. Figure 5 PDK2 Phosphorylates PARL and Regulates β Cleavage
(A) Overexpression of wild-type (WT) PDK2 inhibits β cleavage. In HEK293 cells that overexpress PARL, co-expression of WT PDK2 reduced β cleavage significantly (lane 2). Co-expression of a catalytic inactive mutant N255A PDK2 did not alter β cleavage levels (lane 3).
(B) Quantification of the β cleavage level in each sample in (A).
(C) PDK2 knockdown (KD) induces β cleavage. Stable PDK2 KD and control HEK293 cells were transfected with WT PARL. Relative to untransfected cells, PDK2 KD results in an increase in β cleavage (lane 2). This increase in β cleavage was attenuated by co-transfecting WT (lane 3) but not the catalytic inactive mutant N255A PDK2 (lane 4). PDK2 knockdown and overexpression were confirmed by WB using PDK2 antibody. Mitochondrial protein HSP60 was used as the loading control.
(D) Quantification of the β cleavage level in each sample in (C).
(E) PDK2 inhibition and KD inhibits PARL phosphorylation and induces β cleavage in SH-SY5Y cells. IP was performed on cell lysates from SH-SY5Y cells that were treated with DMSO and DCA and stable PDK2 KD SH-SY5Y cells. PARL phosphorylation status was monitored by WB using phospho-serine/threonine antibody. DCA treatment and PDK2 KD induce β cleavage (second-from-the-top panel) and dramatically reduce the level of phosphorylated PARL relative to untreated cells (top panel), without altering cellular phosphorylation status (bottom panel). PDK2 KD was confirmed by WB using PDK2 antibody. The mitochondrial protein HSP60 was used as loading control.
Results in (B) and (D) were normalized to the control. ∗∗∗p < See also Figures S3–S5.
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8. Figure 6 PDK2 Overexpression Inhibits PINK1/PARKIN-Mediated Mitophagy
(A) Overexpression of wild-type (WT) PDK2 inhibits PARKIN recruitment to damaged mitochondria. HeLa cells were transfected with GFP-PARKIN, and WT and catalytic inactive PDK2 mutant (N255A), and then treated with 20 μM CCCP for 1 hr. PARKIN cellular localization was analyzed by immunofluorescence microscopy with reference to the mitochondrial protein HSP60.
(B) Percentage mitochondrial PARKIN localization in each condition. Overexpression of WT PDK2 inhibits PARKIN’s recruitment to damaged mitochondria, while overexpression of N255A PDK2 mutant did not alter PARKIN localization relative to the control cells. 100 cells were counted per experiment.
(C) PDK2 overexpression delays PARKIN-dependent Mitofusin (MFN) 2 degradation through inhibiting β cleavage. HeLa cells that express PARKIN were transfected with WT PDK2, N255A PDK2, WT PARL, and phosphorylation-resistant PARL mutant (AAA PARL) as indicated; empty pGCA vector was used as control. Transfected cells were treated with 20 μM CCCP at four time points, and the level of MFN2 at each time point was monitored by WB. Relative to the control, overexpression of WT PDK2 but not N255A PDK2 delayed the degradation of MFN2. Co-expression of WT PARL with WT PDK2 did not restore normal MFN2-processing pattern. MFN2-processing pattern was restored with the co-expression of the phosphorylation-resistant AAA PARL. Expression of the different PDK2 and PARL constructs were confirmed by WB. Actin was used as loading control.
(D) Comparison of the MFN2 level at 0- and 15-min CCCP treatment in (C). 15-min CCCP treatment resulted in a significant decrease in MFN2 protein level in control cells. Overexpression of WT PDK2, but not N255A PDK2, stabilized the MFN2 level after 15-min CCCP treatment. Co-expression of WT PARL with WT PDK2 did not significantly reduced MFN2 after 15-min CCCP treatment, while co-expression of a phosphorylation PARL mutant (AAA PARL) lead to significant reduction of the MFN2 level. Results were normalized to the 0-min CCCP treatment in each condition. ∗p < 0.01, ∗∗∗p < Scale bar, 6 μM.
See also Figure S6.
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9. Figure 7 PDK2 Modulates PINK1/PARKIN-Mediated Mitophagy
(A) PDK2 KD stimulates PARKIN mitochondrial recruitment in HeLa cells. Control and PDK2 KD cells were transfected with GFP-PARKIN and treated with 20 μM CCCP for 60 min. Percentage of mitochondrial localized PARKIN at 0- and 15-min CCCP treatment was determined by immunofluorescence microscopy. A significant increase in the percentage of mitochondrial localized PARKIN was observed in PDK2 KD cells in compassion to controls at 0 and 15 min of CCCP treatment.
(B) PDK2 KD promotes PARKIN-dependent Mitofusin (MFN) 2 degradation. Control and PDK2 KD HeLa cells that express GFP-PARKIN treated with 20 μM CCCP for 60 min, and the level of MFN2 at four time points (0, 15, 30, 60 min) was monitored by WB. Relative to the controls, PDK2 KD does not alter the degradation of MFN2 after 60-min CCCP treatment; however, PDK2 KD promotes MFN2 ubiquitination at early time points of CCCP treatment. Actin was used as loading control.
(C) The ratio between ubiquitinated (Ub) MFN2 to MFN2 at 0- and 15-min CCCP treatment in (B). A significant increase in Ub MFN2 to MFN2 ratio was observed between control and PDK2 KD cells. ∗p < 0.01, ∗∗p < See also Figure S7.
(D) In healthy mitochondria, PARL is phosphorylated by PDK2. Phosphorylation of the PARL N terminus inhibits β cleavage, keeping PARL in the fully catalytically active full-length form. PINK1 is imported by the TOM/TIM complex and undergoes a series of processing events, including proteolytic cleavage by PARL. PARL-dependent cleavage generates a cleaved form of PINK1, which is retro-translocated to the cytosol and is rapidly degraded in a proteasome-dependent manner.
(E) As mitochondrial damage accumulates, the activity of components in electron transfer chain is compromised, resulting in ATP depletion. ATP depletion reduces PDK2 activity and therefore reduces the level of phosphorylated PARL. PARL dephosphorylation promotes β cleavage, which increases the production of pß peptide and reduces PARL activity to PINK1 by reducing FL PARL to the catalytically less active PACT. pß translocates to the nucleus and activates the genes that are responsible for mitochondrial biogenesis and metabolism. Reduced PARL activity impairs the proteolytic processing of imported PINK1 and contributes to the stabilization of PINK1 on the outer mitochondrial membrane (OMM). OMM localized PINK1 phosphorylates and activates PARKIN, which, in turn, ubiquitinates its substrates. Phosphorylation of ubiquitin on PARKIN substrates by PINK1 further promotes the mitochondrial recruitment of more cytosolic PARKIN. Escalating mitochondrial damage will eventually disrupt the mitochondrial protein import machinery and promotes further stabilization of PINK1 on OMM and PARKIN mitochondrial recruitment and the removal of damaged mitochondria via mitophagy.
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