Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region

Abstract

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and CD spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

Keywords: IDPR; degradation; mitochondria; mitophagy; protein complex; ubiquitylation.

Figure 1

CLEC16A is predicted to contain an internal intrinsically disordered protein region by complementary in silico analyses. A, structural prediction of CLEC16A using AlphaFold. Model confidence is indicated by the per-residue predicted local distance difference test (pLDDT) from 0 to 100. Very high: pLDDT > 90, High: 90 > pLDDT > 70, Low: 70 > pLDDT > 50, Very low: pLDDT < 50. A low pLDDT score is a competitive predictor of protein disorder (22, 23). B, disorder probability of human and mouse CLEC16A from IUPred2. Putative IDPRs were defined with a probability threshold of >0.5. C, residues of the mouse CLEC16A putative internal IDPR (AA 347–472), with significantly enriched residues identified in panel (D) highlighted (lysine, K, blue; glutamic acid, E, red). D, residue composition bias of the mouse CLEC16A internal IDPR (AA 347–472) generated with Composition. Profiler, comparing residue enrichment of the CLEC16A internal IDPR versus SWISS-PROT 51 database. Significantly enriched lysine (blue) and glutamic acid (red) residues are highlighted. ∗p < 0.05. IDPR, intrinsically disordered protein region.

Figure 2

The internal IDPR contains lysine residues that are essential for CLEC16A turnover. A, schematic of C-terminal Flag epitope-tagged constructs encoding WT full-length CLEC16A, CLEC16A with the internal IDPR residues randomly shuffled (Shuffle IDPR), or CLEC16A lacking the internal IDPR (ΔIDPR). B, representative anti-Flag Western blot (WB) of WT, shuffle IDPR, and ΔIDPR CLEC16A after transfection in HEK293T cells (n = 3/group). C, representative WB of Flag-CLEC16A levels from 293T cells transfected with WT, Shuffle IDPR, or ΔIDPR CLEC16A following treatment with 300 μg/ml cycloheximide for 0 to 6 h. Densitometry represents percent change (%) from basal levels normalized to Cyclophilin B (Cyc B) loading control (n = 4/group). D, predicted ubiquitination of CLEC16A residues, generated by UbPred. E, representative Flag WB of WT, Shuffle IDPR, Shuffle IDPR retain K, IDPR K-to-R, and ΔIDPR CLEC16A following transfection in 293T cells. Densitometry represents fold change in protein levels relative to WT CLEC16A, normalized to Cyc B. No significant differences were found between CLEC16A Shuffle IDPR, IDPR K-to-R, and ΔIDPR (n = 3–4/group). F, representative WB of Flag-CLEC16A levels from 293T cells transfected with WT, IDPR1 K-to-R, and Shuffle IDPR retain K CLEC16A following treatment with 300 μg/ml cycloheximide for 0 to 6 h. Densitometry represents % change from basal levels normalized to Cyc B. n = 3/group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. IDPR, intrinsically disordered protein region.

Figure 3

The CLEC16A internal IDPR is not a site of self-ubiquitination but is required for ubiquitin ligase activity.A, in vitro ubiquitination assay with recombinant CLEC16A-6xHis-Flag WT, ΔIDPR, or IDPR KR. Representative WB and immunoprecipitation (IP) following incubation with ATP, HA-Ubiquitin, E1, and the E2 conjugating enzyme UBE2D3 at 37 °C for 1 h. n = 3/group. B, in vitro ubiquitination assay with recombinant CLEC16A-6xHis-Flag WT and Shuffle IDPR. Representative WB and IP following incubation with ATP, HA-Ubiquitin, E1, and the E2 conjugating enzyme UBE2D3 at 37 °C for 1 h (n = 3/group). IDPR, intrinsically disordered protein region.

Figure 4

RNF41-mediated turnover of CLEC16A depends on lysine residues and their sequence positioning within the internal IDPR of CLEC16A. A, representative WB of Flag-CLEC16A and either HA-EV, HA-RNF41, or HA-CSHQ RNF41 (ligase-dead mutant) transfected into HEK293T cells and treated with cycloheximide (CHX; 300 μg/ml) for 0 to 6 h. n = 3/group. B, representative WB of cell-based ubiquitination assay with overexpressed Flag-tagged CLEC16A and MYC-Ubiquitin cotransfected with either HA-EV, HARNF41, or HA-CSHQ RNF41 performed in HEK293T. Cells were treated with 10 μM MG132 for 12 h (n = 3/group). C, schematic of proposed reciprocal regulation between CLEC16A and RNF41. D, representative Flag WB of WT, Shuffle IDPR, Shuffle IDPR retain K, IDPR K-to-R, and ΔIDPR CLEC16A cotransfected with HA-EV or HA-RNF41 in HEK293T cells. Densitometry represents fold change in protein levels per construct following the addition of RNF41, normalized to Cyc B. No significant differences were found between CLEC16A Shuffle IDPR, Shuffle IDPR Retain K, IDPR K-to-R, and ΔIDPR following the addition of RNF41 (n = 3–6/group). E, representative Flag WB of WT and ΔIDPR CLEC16A cotransfected with HA-EV, HA-RNF41, or HA-dnRNF41 in HEK293T cells. (n = 3/group). ∗∗∗p < 0.001. IDPR, intrinsically disordered protein region.

Figure 5

The CLEC16A internal IDPR promotes the assembly of the CLEC16A–RNF41–USP8 mitophagy complex. A, representative WBs following IP for Flag-CLEC16A and MYC-Ubiquitin from HEK293T cells transfected with plasmids encoding HA-RNF41, MYC-Ubiquitin, and either Flag-EV, WT, Shuffle IDPR, or ΔIDPR CLEC16A. Shuffle IDPR and ΔIDPR CLEC16A plasmids were transfected at 0.2× that of WT CLEC16A (supplemented with Flag-EV vector to equalize the amount of transfected DNA) to normalize protein levels to that of WT CLEC16A (n = 3/group). B, representative WB following IP for HA-RNF41 in HEK293T cells transfected with HA-RNF41, GFP-USP8, and either Flag-EV, WT (1×), Shuffle IDPR (0.2×), or ΔIDPR (0.2×) CLEC16A. Shuffle IDPR and ΔIDPR CLEC16A plasmids were transfected at 0.2× that of WT CLEC16A (supplemented with Flag-EV vector to equalize the amount of transfected DNA) to normalize protein levels to that of WT CLEC16A (n = 3/group). C, representative WB following IP for Flag-CLEC16A in HEK293T cells transfected with HA-RNF41 and either Flag-EV, WT CLEC16A, or CLEC16A IDPR1 only (AA 347–472) (n = 3/group). IDPR, intrinsically disordered protein region.

Figure 6

Experimental confirmation of the CLEC16A internal IDPR using biochemical and biophysical approaches. A, representative WB in HEK293T cells transfected with Flag-CLEC16A constructs (full-length CLEC16A, CLEC16A internal IDPR only (AA 347–472), or CLEC16A ΔIDPR). Beta actin is included as a loading control (n = 3/group). B, ¹H-¹⁵ N HSQC spectra of recombinant CLEC16A internal IDPR (AA 347–472). Spectra clusters near ¹H 8 ppm, consistent with being an IDPR. C, CD spectra of recombinant full-length (WT) mouse CLEC16A. Line represents the average of three reads. Reads were taken of sample at 25 °C (black solid line), then after heating to 90 °C (red line), then after cooling back to 25 °C (black dashed line). Spectra indicates secondary structure is present. D, CD spectra of recombinant CLEC16A internal IDPR (AA 347–472), performed as noted in panel (C). Spectra indicate that secondary structure elements are absent, consistent with an IDPR. HSQC, heteronuclear single quantum coherence; IDPR, intrinsically disordered protein region.

Figure 7

Proposed model for CLEC16A IDPRs’ contributions to its molecular functions and reciprocal regulation by RNF41. The C-terminal IDPR of CLEC16A is known to enhance CLEC16A stability by preventing self-ubiquitination, whereas the functions of the internal IDPR were unknown until the present work. The internal IDPR is critical for the enzymatic function and molecular interactions of CLEC16A. This internal IDPR regulates CLEC16A turnover and is the site by which RNF41 acts to destabilize CLEC16A. The internal IDPR of CLEC16A is also required for its E3 ligase activity, and the loss of this disordered region impairs both self-ubiquitination and the ability of CLEC16A to ubiquitinate RNF41. The internal IDPR binds to RNF41 and mediates the assembly of the CLEC16A–RNF41–USP8 mitophagy complex. IDPR, intrinsically disordered protein region.