Galectins and TRIMs directly interact and orchestrate autophagic response to endomembrane damage

ABSTRACT

Macroautophagy/autophagy is a homeostatic process delivering cytoplasmic targets, including damaged organelles, to lysosomes for degradation; however, it is not completely understood how compromised endomembranes are recognized by the autophagic apparatus. We have described previously that the TRIM family of proteins act as receptors for selective autophagy. In this study we uncovered the property of TRIMs to directly interact with members of the family of cytosolic lectins termed galectins. Galectins patrol the cytoplasm and recognize compromised membranes. We show that TRIM16 uses LGALS3 (galectin 3) to detect damaged lysosomes and phagosomes. TRIM16 assembles the core autophagic machinery and is found in protein complexes with MTOR and TFEB, thus regulating their activity to set in motion endomembrane quality control. The TRIM16-LGALS3 system plays a key role in autophagic homeostasis of lysosomes and in the control of Mycobacterium tuberculosis in vivo.

Galectins are a family of cytosolic lectins known since 2001 to associate with phagosomes containing inanimate objects or bacteria. Lately, they have been reported as markers of lysosomal damage and the ensuing autophagic response. In the study covered here we tested whether galectins can interact with the newly described family of TRIM-class autophagic receptors. We found, much to our surprise, that most of the TRIMs and galectins tested directly interact, which was an unknown feature of either of the families of proteins. A majority of the TRIMs tested interact directly with LGALS3 except for TRIM16, TRIM21, TRIM55 and TRIM56. However, TRIM16 did interact with LGALS3 when ULK1 was added to GST affinity isolation assays. We also found in an siRNA knockdown screen of all human TRIMs that TRIM16 is important for formation of ubiquitin and LC3 puncta in response to lysosomal damage caused by the lumenal buildup of membrane-damaging polymers, such as upon exposure to Leu-Leu-OMe (LLOMe). A CRISPR knockout of TRIM16 in HeLa cells showed significant reduction in LC3 puncta and nearly abrogated ubiquitin puncta in response to LLOMe.

LGALS3 was reported in 2002 to be associated with mycobacterial phagosomes. However, LGALS3 has been reported in comparative tests with LGALS8 in Salmonella-infected cells as not being important in controlling intracellular bacteria. When we tested the significance of LGALS3 using transgenic knockout mice (B6.Cg-Lgals3^m1Poi/J), we found that LGALS3 is important for protection against M. tuberculosis (Mtb) in vivo in models of acute (early) and chronic (long-term) tuberculosis infection. We next found that both LGALS3 and its interacting partner, TRIM16, localize to Mtb phagosomes in infected macrophages, but only when a strain of Mtb capable of perforating the phagosome is used. LGALS3 is required for ubiquitination of Mtb phagosomes, and LGALS3, TRIM16 and ATG16L1 are all required for translocation of Mtb to compartments with autolysosomal properties and for suppressing M. tuberculosis survival in infected macrophages.

TRIM16 interacts with the key autophagy regulators ULK1 and BECN1 and stabilizes them. TRIM16, known to auto-ubiquitinate, also promotes K63 ubiquitination of ULK1 and BECN1 directly or by recruiting additional E3 ligases such as CUL4A (cullin 4A), as CUL4A is found in protein complexes with TRIM16. ATG16L1 is also found in protein complexes with TRIM16, and TRIM16-ATG16L1 association is enhanced in cells treated with LLOMe. Previous studies predicted the existence of an unidentified factor “X” recruiting ATG16L1 to damaged endomembranes (in addition to the ATG16L1 interactions with RB1CC1/FIP200 and ubiquitin), and our physical mapping of the TRIM16 binding site on ATG16L1 indicates that TRIM16 may indeed be this postulated missing factor “X.”

The cells with TRIM16 knocked out by CRISPR (TRIM16^KO) display chronically elevated lysosomal cellular content (i.e., increased number of LAMP2 profiles) of reduced quality (i.e., decreased acidification as assessed by LysoTracker staining). The simplest explanation for this observation could be that lysosomes in the absence of TRIM16 do not undergo efficient autophagic homeostasis. However, we found that a more active mechanism was at play. We found that TRIM16 affects nuclear translocation of TFEB, a transcriptional factor that activates lysosomal biogenesis and increases transcription of several key autophagy factors. TFEB is retained in the cytoplasm by phosphorylation, with lysosomally-located MTOR being one of the key kinases locking TFEB in this location. To translocate to the nucleus, TFEB is dephosphorylated by the PPP3/calcineurin phosphatase. We found that MTOR activity is inhibited in cells subjected to LLOMe treatment. TRIM16 is furthermore found in protein complexes with the MTOR inhibitor DEPTOR, whereas LLOMe treatment stabilizes DEPTOR. The stability of DEPTOR is regulated by CUL5 (cullin 5), and CUL5 is detected in protein complexes with TRIM16. RRAG GTPases control lysosomal localization of MTOR and are important for its activation by amino acids. We found TRIM16 in complexes with RRAGB and RRAGD, consistent with overall regulatory effects of TRIM16 on MTOR. We furthermore found that TFEB is found in TRIM16 complexes, and that TRIM16 and the TFEB phosphatase PPP3 associate through a specific region on TRIM16. Thus, TRIM16 controls MTOR activity and nuclear/cytoplasmic localization of TFEB.

In conclusion, our findings indicate a broader utilization of galectins in combination with TRIMs during autophagic responses to endomembrane damage. Our study furthermore delineates the sequence of events in the very initial recognition of lysosomal or phagosomal damage and how that orchestrates autophagic response, and uncovers that the same system interacts with MTOR and TFEB and controls their function. Thus, the LGALS3-TRIM16 pair orchestrates both the immediate autophagic response and the long-term adaptation to lysosomal damage. We propose that galectin-TRIM pairs play similar roles in autophagic homeostasis of other endomembranes.