Ribosome Profiling and Mass Spectrometry Reveal Widespread Mitochondrial Translation Defects in a Striatal Cell Model of Huntington Disease.

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive deficits, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo-Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded oxidative phosphorylation mRNAs (mt-Nd1, mt-Nd2, mt-Nd4, mt-Nd4l, mt-Nd5, mt-Nd6, mt-Co1, mt-Cytb, and mt-ATP8). We also applied tandem mass tag-based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein abundance of mitochondria-related genes in HD.

SUMO modifies GßL and mediates mTOR signaling.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.