TRIM proteins in autophagy: A study of TRIM32 and TRIM27

Abstract

<p>TRIM proteins are ubiquitin E3 ligases known to be involved in a number of different processes in the cells, such as intracellular signaling, transcription, autophagy, innate immunity, cell cycle progression and DNA repair. Mutations and dysregulation of TRIM proteins are implicated in many diseases, and a number of TRIMs are described as both tumor suppressors and oncogenes. Furthermore, several TRIM proteins are found to have roles in autophagy. <p>We identified TRIM32 as a potential autophagic substrate in a double- tag screen including 22 different TRIM proteins. We found that the autophagic degradation of TRIM32 was selective, and that TRIM32 interacted directly with the autophagy receptors p62 and NDP52, either of which was sufficient for directing TRIM32 to lysosomal degradation. Conversely, we identified both p62 and NDP52 to be TRIM32 substrates. Mutations in the Cterminal region of TRIM32 causes limb girdle muscular dystrophy 2H (LGMD2H). The LGMD2H disease mutants were unable to ubiquitylate p62 and NDP52, and unable to undergo autophagic degradation. Moreover, we showed that reintroduction of TRIM32 in TRIM32 KO cells decreased the protein level of the sequestosome-like receptors (SLRs), while reintroduction of TRIM32 LGMD2H disease mutant did not. Furthermore, we found TRIM32 expression in TRIM32 KO cells to enhance ULK1 stability and TBK1 autophosphorylation, while the LGMD2H mutant did not. The positive effect of TRIM32 on selective autophagy was supported by the finding that mitophagy was reduced in TRIM32 KO cells, while it was restored when TRIM32 was reintroduced. These findings suggest that the pathogenic effect of the LGMD2H mutation at least partially may be due to dysregulation of selective autophagy receptors, affecting the protein homeostasis in muscle cells. <p>We also identified TRIM27 as an autophagy substrate. Similarly as TRIM32, TRIM27 interacted with p62 and NDP52, and its autophagic degradation was dependent on ATG7 and the SLRs. TRIM27 impaired the normal autophagic response of p62 and LC3B when the cells were stressed with plasmid transfection or mimicking of virus transfection. Interestingly, TRIM27 mRNA was significantly upregulated in breast cancer tissues compared to normal tissue, and the expression of TRIM27 in various breast cancer cell lines was inversely correlated with LC3B expression. These results may reveal a link between TRIM27 and dysregulated autophagy in breast cancer.

TRIM proteiner er ubiquitin E3 ligaser med roller i mange forskjellige cellulære prosesser. Mutasjoner og feilregulering av TRIM proteiner er implisert i patologiske tilstander som immunsvikt og kreft. TRIM proteiner er vist å har roller i den cellulære renovasjonsprosessen autofagi. Her identifiserte vi TRIM32 som et potensielt autofagisubstrat i en studie av 22 ulike TRIM proteiner. TRIM32 er uttrykt i alle vev, og mutasjoner i TRIM32 er assosiert med sykdommene Bardet-Biedl-syndrome 11 (BBS11) og Limb-Girdle-muscular dystrophy 2H (LGMD2H). Vi viser at TRIM32 blir degradert ved makroautofagi, og avslører en gjensidig regulering av TRIM32 og autofagireseptorene p62 og NDP52. p62 og NDP52 dirigerte autofagisk degradering av TRIM32, mens TRIM32 ubiquitylerte p62 og NDP52 og dermed påvirket deres autofagiske aktivitet. Våre analyser viser at LGMD2H sykdomsmutantene av TRIM32 ikke er i stand til å regulere autofagireseptorene eller til selv å bli degradert via autofagi. Vi fant også at TRIM32, men ikke LGMD2H sykdomsmutanten, stabiliserte ULK1 proteinet som er viktig for initiering av autofagiprosessen, og fasiliterte degradering av ødelagte mitokondrier. Disse resultatene bidrar til bedre forståelse av den molekylære mekanismen for den patologiske effekten av LGMD2H. Vi identifiserte også TRIM27 som et autofagisubstrat. TRIM27 er et onkogen, oppregulert i flere typer kreft og har en rolle i regulering av medfødt immunitet. Vi viser at TRIM27 binder til p62 og NDP52, og at p62 og NDP52 kan dirigere TRIM27 til autofagisk degradering. Vi fant at TRIM27 kan ha en regulatorisk rolle på autofagiprosessen når den medfødte immunresponsen ble trigget. Våre studier viser at TRIM27 hemmer celleproliferering mens den fremmer cellemigrasjon, noe som kjennetegner invasive kreftceller. TRIM27 mRNA er oppregulert i brystkreftvev. Vi fant TRIM27 og autofagimarkøren LC3B å være dysregulert og invers korrelert i forskjellige typer brystkreftcellelinjer. Disse cellelinjene representerer dermed gode modellsystemer for å forstå TRIM27 og autofagi sin rolle i brystkreft.