Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1

Abstract

The ubiquitin C-terminal hydrolase UCH-L1 (PGP9.5) comprises >1% of total brain protein but is almost absent from other tissues [Wilkinson, K. D., et al. (1989) Science 246, 670–673]. Mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from Parkinson's disease [Leroy, E., et al. (1998) Nature 395, 451–452; Maraganore, D. M., et al. (1999) Neurology 53, 1858–1860]. Abnormal overexpression of UCH-L1 has been shown to correlate with several forms of cancer [Hibi, K., et al. (1998) Cancer Res. 58, 5690–5694]. Because the amino acid sequence of UCH-L1 is similar to that of other ubiquitin C-terminal hydrolases, including the ubiquitously expressed UCH-L3, which appear to be unconnected to neurodegenerative disease, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable importance. We have determined the three-dimensional structure of human UCH-L1 at 2.4-Å resolution by x-ray crystallography. The overall fold resembles that of other ubiquitin hydrolases, including UCH-L3, but there are a number of significant differences. In particular, the geometry of the catalytic residues in the active site of UCH-L1 is distorted in such a way that the hydrolytic activity would appear to be impossible without substrate induced conformational rearrangements.

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

“…To gain further insight into the role of UCHL1 in neurodegeneration, we examined the structural implications and functional consequences of the GLU7ALA mutation. The tertiary structure of UCHL1 resembles that of the papain family (12) and is characterized by the presence of a crossover loop, termed L8, that spans and thereby restricts access to the catalytic cleft that consists of the histidine-cysteine-aspartic acid triad (12,13) (Fig. 2A).…”

“…2A). In UCHL1, the L8 loop is short, thus requiring the ubiquitin substrate to tunnel underneath it to achieve proteolysis; residue E7 is located directly at the threshold of this tunnel (12,13). In the complex of UCHL1 and the ubiquitin substrate mimic, ubiquitin vinyl methyl ester (UbVMe) (13), GLU7 is involved in a hydrogen-bonding network that interacts both with the ubiquitin substrate mimic and the L8 loop ( Fig.…”

“…We next analyzed the effects of the GLU7ALA mutation on ubiquitin binding and C-terminal hydrolase enzymatic activity. In Escherichia coli, we expressed and purified WT UCHL1 (UCHL1 WT ) and mutants UCHL1 GLU7ALA , UCHL1 ILE93MET , and UCHL1 CYS90SER (a cysteine to serine substitution in position 90), which carries a mutation in the active cleft consisting of the abovementioned triad and was previously shown to interfere with the catalytic activity (12)(13)(14). First, the binding of ubiquitin to these purified proteins was measured using isothermal titration calorimetry, from which the K d values for ubiquitin binding to these proteins were calculated ( Fig.…”

Membrane-associated farnesylated UCH-L1 promotes α-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease

“…The discovery and characterization of a membrane-associated and farnesylated form of UCH-L1 provide a new target for further investigation of UCH-L1's role in these phenomena. Membrane association of UCH-L1 may be important to facilitate its interaction with other membrane-associated proteins, such as Lamp-2A (38), to promote its oligomerization (26,28), and/or to induce enzymatic activity (22). Although multiple forms of UCH-L1 have been identified in cell culture and in tissue (16,(55)(56)(57), the possibility that one could be farnesylated had not been entertained previously (58).…”

“…We report here an additional modification that may significantly have an impact on its enzymatic and/or binding activities: UCH-L1 is farnesylated and associated with neuronal membranes, including the endoplasmic reticulum. This modification is not present in UCH-L3, which appears to serve different biochemical and biological roles than UCH-L1 (22)(23)(24)(25)(26)(27)(28)(29). The amount of membrane-associated UCH-L1 (UCH-L1 M ) is linked to intracellular ␣-synuclein levels and neurotoxicity.…”