TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1

Abstract

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Keywords: FIP200; FIR; LIR; Selective autophagy; TAX1BP1; TBK1; TNIP1; mitophagy; mitophagy regulation.

Fig. 1. Ectopic localization of TNIP1 to the mitochondria induces mitophagy.

(A) Schematic representation of TNIP1 and its 2 identified putative LIR motifs. (B) HeLa cells stably expressing HA-TNIP1 wild type, LIR1 mutant, LIR2 mutant or LIR1+LIR2 double mutant co-immunoprecipitated (co-IP) with purified recombinant GST tagged mATG8 proteins and subjected to immunoblot analysis (IB). (C) Schematization of CID experiment. (D) HeLa cells stably expressing mito-mKeima, FRB-FIS1 and FKBP-GFP-TNIP1 WT or UBAN mutant (D472N) were treated with Rapalog for 24 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (E) HeLa cells stably expressing mito-mKeima, FRB-FIS1 and FKBP-GFP-TNIP1 WT, LIR1 mutant, LIR2 mutant or LIR1&LIR2 double mutant were treated with Rapalog for 24 h and subjected to FACS acquisition. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (F) Top, schematic representation of the TNIP family TNIP1, TNIP2 and TNIP3. Bottom, HeLa cells stably expressing mito-Keima, FRB-FIS1 and FKBP-GFP-TNIP1, FKBP-GFP-TNIP2 or FKBP-GFP-TNIP3 were treated with Rapalog for 24 h and subjected to FACS acquisition. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates.

Fig. 2. IR2 and AHD3 domain of TNIP1 are essential for its role in mitophagy.

(A) Schematic representation of TNIP1 and the AHD mutant constructs. (B) HeLa cells stably expressing mito-mKeima, FRB-FIS1 and FKBP-GFP-TNIP1 Full Length (FL) or FKBP-GFP-TNIP1 mutants ΔAHD1, ΔAHD3 and ΔAHD4 were treated with Rapalog for 24 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (C) HeLa cells stably expressing mito-mKeima, FRB-FIS1 and FKBP-GFP-TNIP1 Full Length, LIR2 mutant, ΔAHD3 or LIR2 mutant and ΔAHD3 were treated with Rapalog for 24 h and subjected to FACS acquisition. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (D) Top, schematic representation of full length TNIP1 or the C-terminal and N-terminal part of TNIP1. Bottom, HeLa cells stably expressing mito-Keima, FRB-FIS1 and FKBP-GFP-TNIP1 full length or FKBP-GFP-TNIP1 N-terminal or C-terminal were treated with Rapalog for 24 h and subjected to FACS acquisition. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (E) HeLa cells stably expressing mito-Keima, FRB-FIS1 and FKBP-GFP-TNIP1 in wild type cells, PentaKO (SQSTM1, NBR1, NDP52, TAX1BP1 and TAX1BP1 KO cells), FIP200 KO, WIPI2KO or A20 KO cells were treated with Rapalog for 24 h and subjected to FACS analysis. Bar graph representing data as mean ± SEM obtained from 3 independent replicates. (F) HeLa cells stably expressing mito-Keima, FRB-FIS1 and FKBP-GFP-ATG16L1, FKBP-GFP-WIPI2 or FKBP-GFP-A20 in wild type cells or TNIP1 KO cells were treated with Rapalog for 24 h and subjected to FACS analysis. Bar graph representing data as mean ± SEM obtained from 3 independent replicates.

Fig. 3. TNIP1 is a negative regulator of selective autophagy.

(A) IB of HeLa cells, HeLa TNIP1 KO cells and HeLa TNIP1 KO rescued with FKBP-GFP-TNIP1. Green arrow, FKBP-GFP-TNIP1 construct. Black arrow, endogenous TNIP1. (B) HeLa cells stably expressing mito-mKeima and HA-Parkin in wild type cells, TNIP1 KO cells or TNIP1 KO cells rescued with FKBP-GFP-TNIP1 construct were treated with Oligomycin and Antimycin (O/A) for 6 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (C) HeLa cells stably expressing mito-mKeima and HA-Parkin in wild type cells, TNIP1 KO cells or TNIP1 KO cells rescued with FKBP-GFP-TNIP1 construct were treated with O/A for 2 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (D) HeLa cells stably expressing mito-mKeima, HA-Parkin and overexpressing FKBP-GFP-TNIP1 wild type, ΔAHD1, ΔAHD3 or ΔAHD4 constructs were treated with O/A for 2 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (E) HeLa TNIP1 KO cells stably expressing mito-mKeima, HA-Parkin and rescued with FKBP-GFP-TNIP1 wild type, LIR2 mutant or ΔAHD3 constructs were treated with O/A for 3 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (F) IB of HeLa cells stably expressing BFP-Parkin and GFP-TNIP1 WT treated for 15 h, 18 h, 21 h or 24 h with O/A. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates.

Fig. 4. The interaction between the AHD3 domain of TNIP1 and the Zinc-Finger domain of TAX1BP1 is necessary for TNIP1’s mitophagy inhibition.

(A) Left, High Confidence Candidate Interaction Proteins (HCIPs) network of HA-TNIP1 following an HA-pull down and mass spectrometry analysis. The size of the bait represents higher Z score interaction. Right, heat map of the interactors with wild type TNIP1 or ΔAHD3 and ΔAHD4 mutants. (B) Co-IP and IB of HEK293T cells stably expressing HA-TNIP1 full length, ΔAHD1, ΔAHD3 and ΔAHD4 constructs or HA-TNIP3 using magnetic HA beads. (C) Top, schematic representation of full length TAX1BP1 and the regions encompassing the coiled-coil domain (CC), SKICH truncation (ΔSKICH) and Zinc-Finger truncation (ΔZinc-Finger) constructs. Bottom, Co-IP and IB of HEK293T cells stably expressing GFP-TNIP1 full length and transiently expressing HA-TAX1BP1 full length, CC, ΔSKITCH and ΔZinc-Finger constructs using magnetic GFP beads. (D) HeLa WT cells or TAX1BP1 KO stably expressing mito-mKeima, HA-Parkin and overexpressing FKBP-GFP-TNIP1 construct were treated with O/A for 3 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (E) Co-IP and IB of HeLa TAX1BP1 KO cells stably expressing HA-TNIP1 full length or the ΔAHD1, ΔAHD3, ΔAHD4 constructs using magnetic HA beads. Ø: no overexpressed construct. * Marks unspecific band.

Fig. 5. TNIP1 binds FIP200 via its LIR motif and to the CLAW domain of FIP200.

(A) Co-IP and IB of HEK293T cells stably expressing HA-TNIP1 full length or the ΔAHD1, ΔAHD3, ΔAHD4 and LIR2 mutant constructs using magnetic HA beads. (B) HeLa wild type, TAX1BP1 KO and LC3/GABARAP 6KO cells stably expressing FRB-FIS1 and FKBP-GFP-TNIP1 WT or LIR2m were treated for 24 h with Rapalog and stained for endogenous FIP200 before immunofluorescence acquisition on a confocal microscope. Quantifications as mean ± SEM of Pearson correlation coefficient representing colocalization between FIP200 and GFP. See Fig S5B for representative images. (C) Top, schematic representation of full length FIP200 and the regions encompassing the N-terminal and C-terminal domains as well as the minimal leucine zipper (LZ) and Claw (CLW) domains. Bottom, Co-IP and IB of HEK293T cells stably expressing GFP-TNIP1 full length and transiently expressing HA-FIP200 full length, N-terminal, C-terminal, LZ and CLW constructs using magnetic GFP beads. The zone in dotted lines was exposed longer to reveal the binding to the CLAW domain (lower part).

Fig. 6. TNIP1 binds FIP200 via its FIR motif and is regulated by a phosphorylation upstream of FIR.

(A) Fluorescence polarization of unmodified and phosphorylated FIR peptides of TNIP1 and CCPG1 binding to the CLAW domain of FIP200 with increasing concentrations of CLAW. Error bars represent the standard deviation measured across 3–6 independent experiments for each condition. (B) Competition of the TNIP1 peptides for the displacement of CCPG1 peptides from binding with the CLAW domain of FIP200. Error bars represent the standard deviation measured across four independent experiments for each condition. (C) HeLa cells stably expressing mito-mKeima, HA-Parkin and overexpressing GFP-TNIP1 WT or AAA constructs were treated with O/A for 4 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates. (D) Co-IP and IB of HeLa cells stably expressing GFP-TNIP1 WT or AAA constructs using magnetic GFP beads. (E) Co-IP and IB of HeLa cells stably expressing HA-TNIP1 AAA mutant with purified recombinant GST tagged mATG8 proteins. (F) Co-IP and IB of HEK293T cells stably expressing HA-TNIP1 treated for 6 h with the TBK1 inhibitor GSK8612 and/or O/A for 4 h using magnetic HA beads. (G) Schematic representation of the proposed model of the molecular function of TNIP1. Upon mitophagy stimulation, autophagy receptors are bound to the ubiquitinated mitochondria, and subsequently recruit the ULK1 complex via FIP200 binding, starting the autophagosome formation. Before autophagosomal closure, TNIP1 is targeted to the autophagosome via LC3/GABARAP binding and later activated by TBK1, increasing its binding affinity for FIP200 and competing with the receptors for FIP200 binding, ultimately resulting in release of FIP200 from the autophagosome. Mitophagy can thus proceed and FIP200 can be recycled at another location on the growing phagophore. In a parallel, separate event, excess of TNIP1 inhibits mitophagy by binding TAX1BP1 via AHD3/ZF domains and TAX1BP1 cannot bind ubiquitinated substrates as its UB-binding domain is occupied by TNIP1, as represented in the graphical abstract.