Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) regulates the level of SMN expression through ubiquitination in primary spinal muscular atrophy fibroblasts.
BACKGROUND
Spinal muscular atrophy (SMA), a lethal hereditary disease caused by mutations of the survival of motor neuron 1 (SMN1) gene, is the leading genetic cause of infant mortality. Its severity directly correlates to the expression level of SMN protein in patients with SMA, but the regulatory mechanisms of SMN protein expression remain incompletely defined. In the present study, we aimed to identify candidate proteins to distinguish SMA fibroblasts from normal fibroblasts.


METHODS
To identify cellular targets regulating the expression of SMN, we initially utilized a proteomics approach combining 2D electrophoresis and LC-MS/MS, wherein the total proteins extracted from type I SMA patients and normal skin fibroblast cells were compared.


RESULTS
Our initial proteomics analysis discovered significant increase of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in type I SMA fibroblasts when compared to normal fibroblasts. Significantly, UCHL1 proteins directly interacted with SMN protein, as determined by immunoprecipitation and immunofluorescence assays in P19 and NSC34 cells. Over-expression of UCHL1 in P19 and NSC34 cells significantly reduced the level of SMN proteins in vivo, and, in fact, purified UCHL1 was shown to be able to enhance, in a dose-dependent manner, the level of ubiquitinated SMN in vitro. Further, inhibition of UCHL1 activity by UCHL1 inhibitor (LDN-57444) increased cellular SMN protein and gems number in the nucleus in NSC34 and SMA skin fibroblasts. The same results were observed in cells with UCHL1-specific knockdown.


CONCLUSIONS
These results suggested that UCHL1 may be a critical regulator in controlling cellular SMN protein turnover, and that it may serve as an attractive therapeutic target for SMA.
Hsu
Shih-Hsien
SH
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Lai
Ming-Chi
MC
Er
Tze-Kiong
TK
Yang
San-Nan
SN
Hung
Chih-Hsing
CH
Tsai
Hsin-Hung
HH
Lin
Yu-Chieh
YC
Chang
Jan-Gowth
JG
Lo
Yi-Ching
YC
Jong
Yuh-Jyh
YJ
D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D009134:Muscular Atrophy, Spinal / Q000378:metabolism* / Q000473:pathology*; D040901:Proteomics; D055532:SMN Complex Proteins / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*; D054875:Ubiquitination
D055532:SMN Complex Proteins; C482860:UCHL1 protein, human; D043222:Ubiquitin Thiolesterase