Optineurin provides a mitophagy contact site for TBK1 activation

Abstract

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase involved in many intracellular processes including innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating autophagy adaptors critical in selective autophagic removal of damaged mitochondria (mitophagy). However, the mechanism by which TBK1 is activated by PINK1/Parkin-mediated mitophagy remains largely unknown. Here, we show that the autophagy adaptor OPTN provides a unique platform for TBK1 activation. The OPTN-ubiquitin and OPTN-autophagy machinery interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact site between damaged mitochondria and the autophagosome formation site. This assembly point serves as a positive feedback loop for TBK1 activation by accelerating hetero-autophosphorylation of the protein. Furthermore, expression of monobodies engineered in this study against OPTN impaired assembly of OPTN at the contact sites as well as the subsequent activation of TBK1 and mitochondrial degradation. Taken together the findings reveal that a positive reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD

“…Our results also highlight OPTN's dominant role in recruiting and activating TBK1 during mitophagy. This is in line with the recent finding that OPTN forms a platform for TBK1 activation from where it engages with TBK1 in a positive feedback loop and observations made with the ALS-causing TBK1-E696K mutant, which has lost its OPTN-binding capacity [39,60,61], but not its binding to NAP1 as we show. Although NDP52 can recruit and activate TBK1 in the absence of other cargo receptors, our findings indicate that when OPTN and NDP52 are co-expressed, mitophagy is accelerated when OPTN can more easily recruit TBK1.…”

“…Our data thus propose an essential role for cargo receptors in locally clustering TBK1 dimers on the mitochondrial surface. This is consistent with a recently proposed model, positing that TBK1 is activated from a local platform of OPTN molecules [60].…”

High-affinity mirror-image monobody targeting MCP-1 generated via TRAP display and chemical protein synthesis

“…As a result, sub-µM KD values of 0.34 and 0.48 µM were calculated for Mb5 and Mb8, respectively (Figure 2B). This result was unexpected for us, as high-affinity binders with nM or sub-nM KD were reproducibly selected via TRAP display in our previous studies 37,38 . This discrepancy might be attributed to the less favorable binding patterns between D-and L-proteins compared to those between L-proteins.…”

“…Previously, we obtained high-affinity monobody clones (sub-nM to nM KD) against several protein targets via TRAP display 37,38 . For the first-round selection against the biotinylated D-MCP-1 4D, we employed the same monobody mRNA library as in the previous study 37 , where G-, S-, W-, and Y-rich random residues were introduced into the BC (8 or 10 residues) and FG (10 or 12 residues) loops of monobody to increase the probability of obtaining high-affinity clones 50 .…”

“…To address this issue, we developed an improved version of mRNA display, called TRAP (transcription−translation coupled with association of puromycin linker) display, in which a polypeptide library conjugated with each mRNA sequence is automatically produced via tandem reactions and used for the isolation of macrocyclic peptide binders with double-digit nM affinities 35 . More recently, a fibronectin type III domain-derived protein scaffold, named monobody 36 , was employed in further improved TRAP display selections to generate binders with strong affinities ranging from sub-nM to single-digit nM against multiple targets including SARS-CoV-2 spike protein, EGFR1, HER2 37 and optineurin 38 .…”