Site-specific quantification of the in vivo UFMylome reveals myosin modification in ALS

Abstract

UFMylation is a ubiquitin-like protein modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins and regulates several cellular processes such as protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate remnant UFMylated peptides and identification by mass spectrometry. We used this approach to identify >200 UFMylation sites from various mouse tissues, revealing extensive modification in skeletal muscle. In vivo knockdown of the E2 ligase, UFC1, followed by enrichment and analysis of remnant UFMylated peptides quantified concomitant down-regulation and validation of a subset of modification sites, particularly myosin UFMylation. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with amyotrophic lateral sclerosis (plwALS). Quantification of UFMylation sites in these biopsies with multiplexed isotopic labeling reveal prominent increases in myosin UFMylation. Our data suggest that in vivo UFMylation is more complex than previously thought.

Keywords: CP: cell biology; UFM1; UFMylation; Ubiquitin Fold Modifier 1; ubiquitin-like modification.

Graphical abstract

Figure 1

Enrichment and site-specific identification of the UFMylome (A) Overview of UFMylation and schematic for the enrichment of VG-modified peptides and identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Analysis of antibody specificity to VG-ε-K- compared to GG-ε-K-containing peptides by ELISA. (C) Number of VG-peptide spectral matches (PSMs) and unique VG-peptides/VG-sites identified by each of the antibody clones or the pooled cocktail. (D and E) Overlap of VG-peptides identified by each antibody clone. (F) Motif enrichment of the amino acids surrounding the UFMylation sites. (G) Overlap of VG-peptides identified by each search algorithm. (H) Anti-UFM1 western blot of various mouse and human cell lines, and various mouse tissues. (I) Number of VG-PSMs and unique VG-peptides/VG-sites identified in each mouse tissue. (J and K) (J) Gene Ontology (GO), and (K) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the UFMylated proteins. (L) STRINGdb analysis of the UFMylated proteins.

Figure 2

Site-specific quantification of the UFMylome identifies a differential signature in human skeletal muscle associated with ALS and specific sites of UFMylation on myosin proteins (A) Anti-UFM1 western blot of mouse extensor digitorum longus (EDL) skeletal muscles treated with recombinant adeno-associated virus serotype 6 (rAAV6) harboring scramble negative control shRNA (L: left leg) or UFC1 shRNA (R: right leg). (B) Volcano plot of quantified VG-peptides from mouse EDL following knockdown of UFC1. (C–E) Annotated MS/MS of VG-containing peptides from (C) Lys1381/1381/1375/1378/1381 on MYH1/2/3/4, (D) Lys975/975/969/972 on MYH1/2/3/4, and (E) 1795/4 on MYH4/8, respectively. Inserts show log2(area under the curve) normalized to total myosin levels. ∗∗q < 0.01; paired Student’s t test with Benjamini-Hochberg FDR. Error bars: SEM. (F) Anti-UFM1 western blot of skeletal muscle biopsies from individuals diagnosed with ALS and age-matched controls. (G) Immunoreactivity densitometry of the entire lane. ∗p < 0.05; unpaired Student’s t test. Error bars: SEM. (H) Volcano plot of quantified VG-peptides from human individuals. (I) VG- and GG-peptides identified on myosin purified from mouse skeletal muscle. (J) Modeling the effects of Lys>Trp substitutions on myosin stability.