Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains

Abstract

Label-free quantitative mass spectrometry techniques were applied to discover proteome and ubiquitinome changes in brain tissue of Huntington's disease mouse model Q175FDN and wild-type mice. Triton X-100 soluble and insoluble fractions were analyzed, revealing differential ubiquitination of wild-type and mutant Huntingtin in both. Cellular processes affected by the disease include vesicular transport, gene expression, translation, catabolic processes and oxidative phosphorylation. DiGly site fold changes with respect to protein fold changes were different between soluble and insoluble fractions.

How Do Post-Translational Modifications Influence the Pathomechanistic Landscape of Huntington’s Disease? A Comprehensive Review

“…Insoluble fractions of both wtHTT and mHTT showed ubiquitination at Lys-6 and Lys-9. Moreover, increased ubiquitination and Lys-48 polyubiquitin linkages were identified in the insoluble fraction [13].…”

“…Post-translational modification of effector proteins in the signal transduction of Huntington's disease (HD). phosphorylation (IKK,CK2,NLK,Akt, SGK,CDK5/PP1,PP2A, PP2B) ↓ mHTT aggregation [11] acetylation (CBP/HDAC1) ↓ formation of fibrillary aggregates, lipid-binding [11,12] ubiquitination ↑/↓ proteosomal degradation [13] SUMOylation (PIAS1, RHES) ↑ escape insoluble aggregate formation, neurotoxicity [14] palmitoylation ↓ inclusion formation [15] myristoylation ↓ pathogenic proteolysis [16] caspase cleavage (caspase-1, -6) ↑ mHTT aggregation [17,18] [19,20] Proteolytic cleavage Caspase-6 (CASP6) palmitoylation ↑ CASP6 activation [17] Apoptotic protease-activating factor 1 (Apaf-1) ubiquitination ↑ regulation of caspase-9 [21] Tau impairment and cytoskeletal alterations Serine/arginine-rich splicing factor-6 (SRSF6, aka SRp55) phosphorylation (Dyrk1A) ↑ faulty splicing of tau [22,23] Tau phosphorylation (CDK5/PP2B) ↑ tau aggregation [24] caspase cleavage (caspase-2) ↑ tau truncation [25] Tubulin acetylation ↓ vesicular transport deficit [26,27] Microtubule-associated protein 1 S (MAPS1) acetylation ↓ mHTT degradation [28] β-adducin phosphorylation (PKA) ↑ dendritic spine destabilization [29] Mitochondrial abnormalities and defects in energy metabolism…”

Strategies to Investigate Ubiquitination in Huntington's Disease

“…A recent global proteome and global ubiquitinome analysis of brain tissues of 40 weeks old homozygous Q175FDN mice that express full-length Htt with 175 polyQ repeats, and Q20 wildtype mice as control, used the diGly remnant of ubiquitin left on protein substrates after trypsin digestion to enrich peptides derived from ubiquitinated proteins using a specific antibody. When label-free quantification was used to compare relative protein and diGly peptide levels between HD and wild-type samples, increased levels of the ubiquitin protein and of ubiquitin K48-diGly peptides were observed in the insoluble fraction of HD mice brains (Sap et al, 2019). A similar result was obtained when HA-tagged ubiquitin mutants were transfected into cells expressing mHtt.…”

“…Furthermore, this technique can be combined with quantitative mass spectrometry approaches, and both qualitative and quantitative differences in ubiquitination can be found between different samples in an unbiased fashion. In this way K6, K9, K132, K804, and K837 were identified as ubiquitination sites of soluble full length Htt in brain lysates of 40 weeks old Q175 mice and wild-type controls (Sap et al, 2019). K6 and K9 were mainly ubiquitinated at the mutant soluble Htt protein, while K132, K804, and K837 were mainly ubiquitinated at the wild-type soluble Htt protein.…”

“…In addition, mutations in various phosphorylation sites located in protease-sensitive domains on the Htt protein affected cellular toxicity, and are thus important functional regions (Arbez et al, 2017). While various studies have mapped numerous phosphorylation and acetylation sites, the number of identified ubiquitination sites in Htt is so far limited to K6, K9, K15, K132, K337, K631, K804, K837, and K2097 (Steffan et al, 2004;Yau et al, 2017;Koyuncu et al, 2018;Sap et al, 2019; Figures 1A,B). Ubiquitination and SUMOylation of the N-terminus of mHtt was found to affect both aggregation and HD pathology in cells although the mechanism is still unclear (Steffan et al, 2004).…”