1. Computational and Systems Biology

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function

  1. Jeffrey Molendijk
  2. Ronnie Blazev
  3. Richard J Mills
  4. Yaan-Kit Ng
  5. Kevin I Watt
  6. Daryn Chau
  7. Paul Gregorevic
  8. Peter J Crouch
  9. James BW Hilton
  10. Leszek Lisowski
  11. Peixiang Zhang
  12. Karen Reue
  13. Aldons J Lusis
  14. James E Hudson
  15. David E James
  16. Marcus M Seldin
  17. Benjamin L Parker  Is a corresponding author
  1. Department of Anatomy and Physiology, University of Melbourne, Australia
  2. Centre for Muscle Research, University of Melbourne, Australia
  3. QIMR Berghofer Medical Research Institute, Australia
  4. Department of Biological Chemistry and Center for Epigenetics and Metabolism, University of California, Irvine, United States
  5. Department of Biochemistry and Pharmacology, University of Melbourne, Australia
  6. Children's Medical Research Institute, University of Sydney, Australia
  7. Military Institute of Medicine, Poland
  8. Department of Human Genetics/Medicine, David Geffen School of Medicine, University of California, Los Angeles, United States
  9. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, United States
  10. Charles Perkins Centre, School of Life and Environmental Science, School of Medical Science, University of Sydney, Australia
https://doi.org/10.7554/eLife.82951
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Cite this article
  1. Jeffrey Molendijk
  2. Ronnie Blazev
  3. Richard J Mills
  4. Yaan-Kit Ng
  5. Kevin I Watt
  6. Daryn Chau
  7. Paul Gregorevic
  8. Peter J Crouch
  9. James BW Hilton
  10. Leszek Lisowski
  11. Peixiang Zhang
  12. Karen Reue
  13. Aldons J Lusis
  14. James E Hudson
  15. David E James
  16. Marcus M Seldin
  17. Benjamin L Parker
(2022)
Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function
eLife 11:e82951.
https://doi.org/10.7554/eLife.82951
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  3. Download .RIS
7 figures, 1 table and 9 additional files

Figures

Figure 1 with 2 supplements
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Proteome-wide systems genetics analysis of the mouse skeletal muscle proteome.

(A) Overview of the experimental design. (B) Number of proteins identified. (C) Intra- and inter-strain coefficient of variation. (D) Protein-quantitative trait loci (pQTL) Manhattan plot. (E) pQTL …

Figure 1—figure supplement 1
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Hybrid Mouse Diversity Panel (HMDP) mouse sample dendrogram.

Dendrogram of HMDP mouse samples based on Euclidean distance and Ward’s clustering criterion (n=161).

Figure 1—figure supplement 2
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EPHX1 Arg338Cys mutation analysis.

(A) EPHX1 AlphaFold structure. Structure zoom-in highlighting Arg338Cys mutation and previously identified mutations with adverse health outcomes. (B) Arg338Cys is highlighted in yellow, the …

Figure 2
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Protein and phenotype quantitative trait locus (QTL) analysis.

(A) Manhattan plot and genomic location distribution of mol/pheQTLs. (B) Overview of the three-step integrative analysis approach. (C) Mirrored Manhattan plots of MCEE and glucose QTLs. (D) Allelic …

Figure 3
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Proteome-phenotype associations of Qrr1 region on chromosome 1.

(A) Manhattan plot of selected genes located near the Qrr1 region, with corresponding traits. (B) Protein-trait correlation network. (C) Top 10 GeneBass associations from the ‘UK BioBank Assessment …

Figure 4
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Functional screening of skeletal muscle function.

(A) Overview of experimental design. (B) Knockdown efficiency of target proteins (n=4–10). (C) Maximum tetanic force, and (D) % fatigue of rAAV6:shScramble and target proteins. Red: q<0.05; yellow: …

Figure 5
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UFMylation is regulated in atrophy and influences skeletal muscle function.

(A) Western blot and (B) densitometry of UFMylation and BiP chaperone in a gastrocnemius muscle of a mouse model of amyotrophic lateral sclerosis (ALS). (C) Overview of the experimental design. (D) …

Figure 5—source data 1

Zip file containing uncropped western blot image files as Image Lab Documents, tiff files, and a summarized.pdf highlighting the lane identifications, highlighted bands used to create Figure 5A, antibody information, and all densitometry results for each individual sample.

The top corner of each membrane is cut above lane 1.

https://cdn.elifesciences.org/articles/82951/elife-82951-fig5-data1-v2.zip
Figure 5—source data 2

Zip file containing uncropped western blot image files as Image Lab Documents, tiff files, and a summarized.pdf highlighting the lane identifications, highlighted bands used to create Figure 5D, antibody information, and all densitometry results for each individual sample.

The top corner of each membrane is cut above lane 1.

https://cdn.elifesciences.org/articles/82951/elife-82951-fig5-data2-v2.zip
Figure 6
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Characterization of skeletal muscles following UFC1 knockdown.

(A) Representative immunofluorescence microscopy of fiber-type composition in tibialis anterior (TA). Myosin heavy chain isoforms (MYH2, green, type IIa; MYH4, purple, type IIb; MYH1, unstained, …

Figure 6—source data 1

Zip file containing uncropped western blot image files as Image Lab Documents, tiff files, and a summarized.pdf highlighting the lane identifications, highlighted bands used to create Figure 6H, antibody information and all densitometry results for each individual sample.

The top corner of each membrane is cut above lane 1.

https://cdn.elifesciences.org/articles/82951/elife-82951-fig6-data1-v2.zip
Author response image 1
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Comparison of UFC1 abundance and cis-pQTL SNPs in skeletal muscle of several inbred mouse strains.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyRabbit monoclonal anti-UFC1AbcamEPR15014-102 (ab189252)(1:1000)
AntibodyRabbit monoclonal anti-UFSP2AbcamEP13424-49 (ab192597)(1:1000)
AntibodyRabbit monoclonal anti-UFM1AbcamEPR4264(2) (ab109305)(1:1000)
AntibodyRabbit monoclonal antiBiPCell Signaling Technologies3177(1:1000)
AntibodyRabbit monoclonal SQSTM1/p62 (D1Q5S)Cell Signaling Technologies39749(1:1000)
AntibodyRabbit monoclonal K48-linkage Specific Polyubiquitin (D9D5)Cell Signaling Technologies8081(1:1000)
AntibodyDonkey polyclonal Anti-Rabbit-HRPJackson ImmunoResearch711-035-152 (RRID:AB_10015282)(1:10000)
Genetic reagent (Homo sapiens)Human Skeletal MyoblastsLonzaCC-2580 (lot #18TL269121)
Genetic reagent (Homo sapiens)Human embryonic kidney 293 cells expressing SV40 large T antigenATCCCRL-1573
Strain, strain background (Mus musculus)A/JJAXRRID:IMSR_JAX:000646
Strain, strain background (Mus musculus)AXB10/PgnJJAXRRID:IMSR_JAX:001681
Strain, strain background (Mus musculus)AXB13/PgnJJAXRRID:IMSR_JAX:001684
Strain, strain background (Mus musculus)AXB15/PgnJJAXRRID:IMSR_JAX:001685
Strain, strain background (Mus musculus)AXB19a/PgnJJAXRRID:IMSR_JAX:001686
Strain, strain background (Mus musculus)AXB4/PgnJJAXRRID:IMSR_JAX:001676
Strain, strain background (Mus musculus)AXB8/PgnJJAXRRID:IMSR_JAX:001679
Strain, strain background (Mus musculus)B6.Cg-Tg(SOD1*G37R)42Dpr/JJAXRRID:IMSR_JAX:008342
Strain, strain background (Mus musculus)BALB/cByJJAXRRID:IMSR_JAX:001026
Strain, strain background (Mus musculus)BTBR T+tf/JNANA
Strain, strain background (Mus musculus)BUB/BnJJAXRRID:IMSR_JAX:000653
Strain, strain background (Mus musculus)BXA12/PgnJJAXRRID:IMSR_JAX:001700
Strain, strain background (Mus musculus)BXA13/PgnJJAXRRID:IMSR_JAX:001826
Strain, strain background (Mus musculus)BXA14/PgnJJAXRRID:IMSR_JAX:001702
Strain, strain background (Mus musculus)BXA16/PgnJJAXRRID:IMSR_JAX:001703
Strain, strain background (Mus musculus)BXA2/PgnJJAXRRID:IMSR_JAX:001693
Strain, strain background (Mus musculus)BXA4/PgnJJAXRRID:IMSR_JAX:001694
Strain, strain background (Mus musculus)BXD100NANA
Strain, strain background (Mus musculus)BXD100/RwwJJAXRRID:IMSR_JAX:007143
Strain, strain background (Mus musculus)BXD12/TyJJAXRRID:IMSR_JAX:000045
Strain, strain background (Mus musculus)BXD14/TyJJAXRRID:IMSR_JAX:000329
Strain, strain background (Mus musculus)BXD19/TyJJAXRRID:IMSR_JAX:000010
Strain, strain background (Mus musculus)BXD21/TyJJAXRRID:IMSR_JAX:000077
Strain, strain background (Mus musculus)BXD22/TyJJAXRRID:IMSR_JAX:000043
Strain, strain background (Mus musculus)BXD27/TyJJAXRRID:IMSR_JAX:000041
Strain, strain background (Mus musculus)BXD28/TyJJAXRRID:IMSR_JAX:000047
Strain, strain background (Mus musculus)BXD29/TyJNANA
Strain, strain background (Mus musculus)BXD31/TyJJAXRRID:IMSR_JAX:000083
Strain, strain background (Mus musculus)BXD32/TyJJAXRRID:IMSR_JAX:000078
Strain, strain background (Mus musculus)BXD33/TyJJAXRRID:IMSR_JAX:003222
Strain, strain background (Mus musculus)BXD34/TyJJAXRRID:IMSR_JAX:003223
Strain, strain background (Mus musculus)BXD39/TyJJAXRRID:IMSR_JAX:003228
Strain, strain background (Mus musculus)BXD40/TyJJAXRRID:IMSR_JAX:003229
Strain, strain background (Mus musculus)BXD44/RwwJJAXRRID:IMSR_JAX:007094
Strain, strain background (Mus musculus)BXD45/RwwJJAXRRID:IMSR_JAX:007096
Strain, strain background (Mus musculus)BXD48/RwwJJAXRRID:IMSR_JAX:007097
Strain, strain background (Mus musculus)BXD48ANANA
Strain, strain background (Mus musculus)BXD5/TyJJAXRRID:IMSR_JAX:000037
Strain, strain background (Mus musculus)BXD50/RwwJJAXRRID:IMSR_JAX:007099
Strain, strain background (Mus musculus)BXD51/RwwJJAXRRID:IMSR_JAX:007100
Strain, strain background (Mus musculus)BXD55/RwwJJAXRRID:IMSR_JAX:007103
Strain, strain background (Mus musculus)BXD60/RwwJJAXRRID:IMSR_JAX:007105
Strain, strain background (Mus musculus)BXD61/RwwJJAXRRID:IMSR_JAX:007106
Strain, strain background (Mus musculus)BXD62/RwwJJAXRRID:IMSR_JAX:007107
Strain, strain background (Mus musculus)BXD63NANA
Strain, strain background (Mus musculus)BXD65NANA
Strain, strain background (Mus musculus)BXD66/RwwJJAXRRID:IMSR_JAX:007111
Strain, strain background (Mus musculus)BXD67/RwwJJAXRRID:IMSR_JAX:007112
Strain, strain background (Mus musculus)BXD68/RwwJJAXRRID:IMSR_JAX:007113
Strain, strain background (Mus musculus)BXD69/RwwJJAXRRID:IMSR_JAX:007114
Strain, strain background (Mus musculus)BXD73/RwwJJAXRRID:IMSR_JAX:007117
Strain, strain background (Mus musculus)BXD75/RwwJJAXRRID:IMSR_JAX:007119
Strain, strain background (Mus musculus)BXD86/RwwJJAXRRID:IMSR_JAX:007129
Strain, strain background (Mus musculus)BXD87/RwwJJAXRRID:IMSR_JAX:007130
Strain, strain background (Mus musculus)BXH10/TyJJAXRRID:IMSR_JAX:000032
Strain, strain background (Mus musculus)BXH14/TyJJAXRRID:IMSR_JAX:000009
Strain, strain background (Mus musculus)BXH8/TyJJAXRRID:IMSR_JAX:000076
Strain, strain background (Mus musculus)C3H/HeJJAXRRID:IMSR_JAX:000659
Strain, strain background (Mus musculus)C57BL/6JJAXRRID:IMSR_JAX:000664
Strain, strain background (Mus musculus)C58/JJAXRRID:IMSR_JAX:000669
Strain, strain background (Mus musculus)CBA/JJAXRRID:IMSR_JAX:000656
Strain, strain background (Mus musculus)CE/JJAXRRID:IMSR_JAX:000657
Strain, strain background (Mus musculus)CXB12/HiAJJAXRRID:IMSR_JAX:001633
Strain, strain background (Mus musculus)CXB2/ByJJAXRRID:IMSR_JAX:000352
Strain, strain background (Mus musculus)DBA/2JJAXRRID:IMSR_JAX:000671
Strain, strain background (Mus musculus)FVB/NJJAXRRID:IMSR_JAX:001800
Strain, strain background (Mus musculus)LG/JJAXRRID:IMSR_JAX:000675
Strain, strain background (Mus musculus)LP/JJAXRRID:IMSR_JAX:000676
Strain, strain background (Mus musculus)MRL/MpJJAXRRID:IMSR_JAX:000486
Strain, strain background (Mus musculus)NON/ShiLtJJAXRRID:IMSR_JAX:002423
Strain, strain background (Mus musculus)NOR/LtJJAXRRID:IMSR_JAX:002050
Strain, strain background (Mus musculus)NZB/BINJJAXRRID:IMSR_JAX:000684
Strain, strain background (Mus musculus)PL/JJAXRRID:IMSR_JAX:000680
Strain, strain background (Mus musculus)SJL/JJAXRRID:IMSR_JAX:000686
Software, algorithmR version 4.1.1R Development Core Team, 2016https://www.R-project.org/
Software, algorithmLimma 3.32.2Ritchie et al., 2015https://bioconductor.org/packages/release/bioc/html/limma.html
Software, algorithmCoffeeProtMolendijk and Parker, 2021ahttps://www.coffeeprot.com
Software, algorithmTeaProtMolendijk et al., 2022https://tea.coffeeprot.com
Software, algorithmMol* (Molstar)Sehnal et al., 2021https://molstar.org/
Software, algorithmWGCNALangfelder and Horvath, 2008https://cran.r-project.org/web/packages/WGCNA/
Software, algorithmColabFold (Alphafold2)Mirdita et al., 2022https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb
Software, algorithmGenebassKarczewski et al., 2022https://app.genebass.org/
Software, algorithmFoldXDelgado et al., 2019http://foldxsuite.crg.eu/
Software, algorithmPROVEANChoi and Chan, 2015http://provean.jcvi.org/index.php
Software, algorithmDynaMutRodrigues et al., 2018http://biosig.unimelb.edu.au/dynamut/

Additional files

Supplementary file 1

Hybrid Mouse Diversity Panel (HMDP) skeletal muscle proteomics.

Proteomics data of gastrocnemius muscle displaying quantification ratios of each sample compared to its corresponding pooled tandem mass tag (TMT) control. PEP: posterior error probability. Related to Figures 1—3, Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp1-v2.xlsx
Supplementary file 2

Hybrid Mouse Diversity Panel (HMDP) skeletal muscle protein-quantitative trait loci (pQTLs).

Protein quantitative trait loci from 161 HMDP cohort mice. Table contains cis-pQTLs (p < 1×10−4) and trans-pQTLs (p < 5 × 10−8), including genomic locations of the single nucleotide polymorphism (SNP) and associated gene. pQTLs are annotated with proxy (cis/trans), intragenic variants, known LD blocks, variant effect, variant impact, and target screen prioritization columns. Related to Figures 1—3, and Figure 1—figure supplement 2.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp2-v2.xlsx
Supplementary file 3

Hybrid Mouse Diversity Panel (HMDP) skeletal muscle pairwise protein-protein correlations.

Protein-protein correlation as determined using biweight midcorrelation. p-Values and q-values derived using the Benjamini-Hochberg procedure, and only positive correlations are shown (cor > 0.3 and q < 0.05). Correlated protein pairs are annotated with protein:protein interactions from the CORUM and BioPlex databases, and subcellar. Related to Figures 1 and 2.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp3-v2.xlsx
Supplementary file 4

Hybrid Mouse Diversity Panel (HMDP) molecular or phenotypic traits.

Summary of traits integrated into the current study including Pubmed ID sources. Related to Figures 2 and 3.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp4-v2.xlsx
Supplementary file 5

Hybrid Mouse Diversity Panel (HMDP) molecular or phenotypic quantitative trait loci (QTLs).

Table contains QTLs (p < 1×10−4) including chromosome, genomic location, including Pubmed ID sources. Related to Figures 2 and 3.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp5-v2.xlsx
Supplementary file 6

Proteomics of skeletal muscle treated with either rAAV6:shScramble or AAV6:shUFC1.

Proteomics of extensor digitorum long (EDL) muscles displaying tandem mass tag (TMT) quantification expressed as Log2(area under the curve). Significance was calculated using paired Student’s t-test with Benjamini-Hochberg FDR. PEP: posterior error probability. Related to Figure 4.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp6-v2.xlsx
Supplementary file 7

shRNA sequences used in the human micro-muscle screen and mouse shUFC1 experiments.

Related to Figure 4.

https://cdn.elifesciences.org/articles/82951/elife-82951-supp7-v2.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/82951/elife-82951-mdarchecklist1-v2.docx
Source data 1

Genebass datasets for UFC1, EPHX1, DUSP23, NIT1, MPZ, BPNT1 and PCP4L1.

https://cdn.elifesciences.org/articles/82951/elife-82951-data1-v2.zip

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