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Then, chloroquine and MG132 were treated to determine which protein degradation pathway is related, and it was confirmed that
Characterization of a novel truncating variant of
TBL1X identified in dilated cardiomyopathy

1
1
Hyeong-Jin Kim , Sun-Ho Lee , Sae-Bom Jeon , Hyoeun Kim , Jungyoon Choi , Jaewon Oh ,
3
1,*
2,*
1
1,2
1,2
Ho-Geun Yoon , Sahng Wook Park , and Seung-Hyun Lee 1,2,†
1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, South Korea
2 Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, South Korea
3 Division of Cardiology, Severance Cardiovascular Hospital, Cardiovascular Research Institute, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, South Korea
Abstract C D In vitro cell-free translation assay E
TBL1X WT
Dilated cardiomyopathy (DCM) is a cardiac disease that causes heart failure and is a leading cause of heart
transplantation. To investigate the relationship between disease and associated genes, whole genome
TBL1X TV
sequencing analysis was performed with a cohort of DCM patients and their family. We found a novel
homozygous nonsense mutation in exon 15 of the transducin β-like 1 X-linked protein (TBL1X). TBL1X is a
subunit of the co-repressor SMRT and plays a role in the cell type differentiation. In our study, we found that Flag-TBL1X WT Ubiquitinated
proteins
TBL1X truncated variant (TBL1X TV ) protein levels were significantly reduced compared to wild-type in H9C2 Flag-TBL1X TV F
cell, although there was no difference in the mRNA levels. In addition, inhibition of proteasome activity by
MG132 effectively rescued the reduced protein level of TBL1X TV . These results suggested that TBL1X
degradation is proteasome-dependent. Also, we identified phosphorylation at serine 470/474 and
sumoylation at Lysine 560 in the truncated region. We investigated whether protein stability was regulated
by post-translational modification. When, restoration of sumoylation to TBL1X TV , the level of protein
expression was increased. Furthermore, TBL1X protein expression levels were decreased in human DCM
heart tissues compared to normal tissues. So, we suggest that TBL1X expression level and protein truncation
are related to the cause of the DCM.
Background Figure 2. (continued)
(C) Protein expression of C-terminus-truncated FLAG-tagged TBL1X (TBL1X TV , lane 3) was confirmed in a immunoblotting
with anti-FLAG antibody. In lane 2, expression of the full-length FLAG-TBL1X protein was shown. (D) (2, 4 lanes) pSG5-
TBL1X WT and TBL1X TV constructs were translation assay in vitro, and products were immunoblotted and reacted with
Dilated cardiomyopathy (DCM) characterized left ventricular dilation associated with systolic FLAG-tag antibody. (1, 3 lanes) pSG5-TBL1X WT , TBL1X TV were transiently transfected into H9C2 cells, and lysates were
dysfunction. Sometimes patients show heart failure, a life-threatening risk, even though they have been immunoblotted and reacted with anti-FLAG antibody. (E) TBL1X TV and TBL1X WT was transfected 293FT cells. TBL1X protein
treated. Other life-threatening risks are arrhythmias and atrioventricular blocks. Inflammatory disorders was immunoprecipitated with M2 FLAG bead. The ubiquitinated products were detected by immunoblotting with anti-HA
or toxic effects from alcohol or drugs cause dilated cardiomyopathy, and genetic factors affected 30-40% antibody. (F) (Left) Representative immunohistochemistry (IHC) staining of ICMP, DCMP patient heart tissues using anti-
TBL1X antibody. Black arrows, nuclei of cardiomyocyte. Scale bar : 50μm. (Right) Bar graph showing quantification of
of patients. About 25~35% of affected individuals have familial forms of the disease. TBL1X level. Quantificationof TBL1X expression by ImageScope. Error bars represent the mean ± SD (n=4, **P <0.005)
Transducin β-like 1 X-linked protein (TBL1X), a transcriptional cofactor, is initially identified as a core
component of the nuclear receptor corepressor (NCoR) complex and also co-immunoprecipitates with
silencing mediator of retinoic acid and thyroid receptor (SMRT). The TBL1X gene has 18 exons, spans
approximately 150 kb of genomic sequence on chromosome Xp22.3. The NCoR complex is responsible A B
for the repression of many different transcription factors. We found a novel homozygous nonsense
mutation c.1316G>A (p.Trp439Ter) in exon 15 of the TBL1X protein. TBL1X protein has three main
domains : LisH domain, F-box-like domain and WD40 repeat domain. Especially, WD40 domain mediated
protein-protein interaction, signal transduction, RNA processing. This mutation site was on WD40 repeat
domain. In addition, we identified phosphorylation at serine 470/474 and sumoylation at Lysine 560 in
the full length TBL1X. So we expected that TBL1X TV form has the protein stability dysfunction.
Previously, Sabrina Rivero et al reported that TBL1X is required for the mesenchymal phenotype of
transformed breast cancer cells. Also Youssef et al reported that TBL1X as a novel therapeutic strategy in
Diffuse large B-cell lymphoma (DLBCL). However, despite the high level of TBL1X protein expression in
the heart, the relationship between TBL1X and related diseases has never been studied in the heart. In
this study, we identified genetic variant in the TBL1X gene, which are likely to be associated with disease
in Dilated cardiomyopathy patient, and confirmed that the stability of the protein was reduced, which C
involved the post-translation modification (PTM) site located in the truncated region.
Results
A B D E
Heart disease Hepatocirrhosis
Hypertension
Diabetes mellitus
C DCM patient Figure 3. Sumoylation at lysine 560 regulates protein stability.
(A) H9C2 cells were transiently transfected with pSG5-TBL1X WT or TBL1X TV constructs. At 24 hours post-transfection, cells
were left untreated (-) or treated with (A) 10uM MG132 for the indicated time points. Whole-cell lysates were prepared
and subjected to immunoblotting using anti-FLAG antibody against p53 and β-actin. (B) 24hr Chloroquine for the indicated
concentrations, immunoblotting using anti-FLAG antibody against LC3II and β-actin. (C) (left) Schematic structures of
truncated TBL1X proteins. Positions of phosphorylation sites and sumoylation site were indicated. (right) Serine residues
at positions 470 and 474 were mutated to alanine, Lysine residue at position 560 was mutated to Arginine. (D) H9C2 cells
were transiently transfected with pSG5-TBL1X WT or TBL1X K560R or TBL1X S470/474A constructs. At 24 hours post-transfection,
cells were left untreated (-) or treated with 10uM MG132 for 12hr. (E) (left) H9C2 cells were transiently transfected with
Figure 1. Mutational characteristics of TBL1X p.W439*. pSG5-TBL1X WT or TBL1X TV , or sumo-tagged TBL1X TV constructs. (right) H9C2 cells were transiently transfected with pSG5-
(A) The Pedigree of DCMP family with TBL1X p.W439*. Black box indicated homozygous affected individual. (B) IGV TBL1X WT or TBL1X K560R or sumo-tagged TBL1X K560R constructs.
snapshot of c.1316G>A, p.Trp439* variant in TBL1X (ChrX: 9,677,677, hg19). Nonsense mutation was found using
next-generation sequencing. (C) Sequence alignment showing point of truncation of TBL1X protein. It was highly
Conclusion
conserved crossing vertebrate, insect and yeast genomes.
A
TBL1X WT In this study, we have identified a novel homozygous nonsense-mutation in TBL1X. As a result of in vitro
TBL1X WT
cell-free translation assay to determine why the protein expression level of TBL1X truncated protein
decreased, it was found that there was a problem in the degradation stage rather than the synthesis stage.
Then, chloroquine and MG132 were treated to determine which protein degradation pathway is related,
TBL1X TV and it was confirmed that the expression level of the truncated protein was particularly increased when
TBL1X TV the activity of proteasome was suppressed. While looking for the reason for the decrease in protein
stability, we confirmed that there was a PTM sites in the truncated region.
As a result, when the K560 where sumoylation occurs was modified, it showed similar protein expression
B Quantitative PCR
1 .5 level to the level of the truncated protein, and when the sumo protein was attached to tne N-term of the
2 .0 Pre-PTC 1 .5 Post-PTC N o n - t r e a t truncated protein, the level of protein expression was restored. Previously, TBL1X was reported to be
R e la tiv e e x p r e s s io n /G A P D H 1 .5 1 .0 0 .5 R e la tiv e e x p r e s s io n /G A P D H R e la tiv e e x p r e s s io n /G A P D H 1 .0 0 .5 1 .0 involved Wnt signaling together with TBLR1, and the reduced protein level by TBL1X TV is expected to
C H X
affect Wnt signaling. Thus, further studies for interaction between TBL1X and Wnt signaling are needed.
0 .5
0 .0
T B L 1 X -W T T B L 1 X -T V 0 .0 0 .0 B L 1 X -W T T B L 1 X -T V References
T
T B L 1 -W T
T B L -T V
Wnt signaling.Mol Cell. 2011;43(2):203-16.
Figure 2. Nonsense variant in the TBL1X escape NMD and produce a truncated protein. 1. Choi HK, Choi KC, Yoo JY, Song M, Ko SJ, Kim CH, et al. Reversible SUMOylation of TBL1-TBLR1 regulates beta-catenin-mediated
(A) Domain structure of TBL1X showing the nonsense mutation introduced a premature stop codon which was 2. Tatsuaki Kurosaki, Maximilian W. Popp & Lynne E. Maquat. Quality and quantity control of gene expression by nonsense-mediated
mRNA decay. Nat Rev Mol Cell Biol. 2019;20(7):406-420.
predicted to truncate the protein within the C-terminal WD40 repeat domain. TBL1X chromatograms of normal 3. Pohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science. 2019;366(6467):818-822.
(TBL1X WT ) and p.W439* (TBL1X TV ). Cytosine in the TBL1X WT was substituted to Thymine in the mutant (red box), 4. John Lynn Jefferies MD, Prof Jeffrey A Towbin MD. Dilated cardiomyopathy. Lancet. 2010;375(9716):752-62
resulting in the change from Tryptophan to a stop codon at amino acid 439. (B) Levels of TBL1X WT and TBL1X TV -PTC 5. Annie Turkieh, Sina Porouchani, Olivia Beseme, Maggy Chwastyniak, Philippe Amouyel, Nicolas Lamblin, et al. Increased clusterin
related mRNAs were also measured by quantitative PCR(qPCR) in the H9C2 cells. Values were expressed as fold levels after myocardial infarction is due to a defect in protein degradation systems activity. Cell Death Dis. 2019;10(8):608
changes (means ± SD, n=3). Cells were treated in the presence of PBS (control) or cycloheximide (100ug/ml) for 2h. 6. Ya-Ting Wang, Jocelyn Chen, Chou-Wei Chang, Jayu Jen, Tzu-Yu Huang, et al. Ubiquitination of tumor suppressor PML regulates
Pre-PTC, Post-PTC means primer binding location. prometastatic and immunosuppressivetumor microenvironment. J Clin Invest. 2017;127(8):2982-2997