The third and most recently identified Parkinson's disease-linked variant of the neuronal protein R-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K R-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K R-synuclein formed insoluble fibrils in Vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K R-synuclein fibrils possess a typical amyloid ultrastructure. E46K R-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of R-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type R-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in Vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of R-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT R-synuclein that are observed in Vitro.Parkinson's disease (PD 1 ) is a progressive neurodegenerative disorder characterized by resting tremor, bradykinesia, rigidity, and postural instability due to the selective loss of dopaminergic neurons within the substantia nigra (2, 3). While nearly all cases of PD are idiopathic, rare forms of autosomal dominant PD have been linked to the point mutations A53T (4), A30P (5), and E46K (1) in R-synuclein, a presynaptic protein believed to be involved in synaptic vesicle trafficking (6,7). Linkage between idiopathic PD and R-synuclein is suggested by the discovery that Lewy bodies (LB), intraneuronal cytoplasmic inclusions in the substantia nigra that are the pathological hallmark of PD, are composed primarily of fibrillar R-synuclein (8).Monomeric R-synuclein is natively unfolded in solution (9), but assumes -sheet character as it aggregates through a series of intermediate, metastable oligomeric states (termed protofibrils) to a stable fibrillar conformation (10). In Vitro studies have shown that both the A53T and A30P mutations in R-synuclein alter the kinetics of fibrillization; the rate is increased for the A53T variant and retarded by the A30P substitution (11,12). However, in both cases the rates of formation of pre...
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.
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-L1 M ) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-L1 M in transfected cells is shown to correlate with the intracellular level of ␣-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-L1 M in cell culture models of ␣-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces ␣-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-L1 M , suggesting that it may negatively regulate the lysosomal degradation of ␣-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies.farnesylation ͉ synuclein
Deubiquitinating enzymes (DUBs) are negative regulators of protein ubiquitination and play an important role in ubiquitindependent processes. Recent studies have found that diverse cellular mechanisms are employed to control the activity of DUBs. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a highly expressed neuronal DUB linked to Parkinson disease; however, little is known about its specific functions or modes of regulation. Here, we demonstrate that UCH-L1 is post-translationally modified by monoubiquitin in cells, at lysine residues near the active site. This modification restricts enzyme activity by preventing binding to ubiquitinated targets, and permanent monoubiquitination, as mimicked by a ubiquitin-UCH-L1 fusion, inhibits UCH-L1 in its capacity to increase free ubiquitin levels in cells. Interestingly, UCH-L1 catalyzes its own deubiquitination in an intramolecular manner, thereby regulating the lifetime of this modification. Our results illustrate monoubiquitination as a reversible regulatory mechanism for DUB activity involving auto-deubiquitination.Protein ubiquitination is a central regulator in numerous cellular processes, including protein degradation, cell cycle progression, and transcriptional regulation (1). The enzymatic system for conjugating ubiquitin to protein substrates is well characterized and results in conjugation of ubiquitin to the ⑀-amino group of lysine residues or in some cases to the N-terminal amino group of target proteins (2, 3). Proteins can be modified by monomeric ubiquitin (termed monoubiquitination) or by ubiquitin chains formed by conjugation of additional ubiquitin molecules to various lysines within ubiquitin (referred to as polyubiquitination). The number and conformation of appended ubiquitin molecules determine the fate of the target protein. While Lys-48-linked polyubiquitin chains typically target proteins for proteasomal degradation, monoubiquitination at one or more lysines triggers a variety of effects depending on the substrate protein, including endocytosis, gene silencing, and DNA repair (4, 5). Our understanding of the many functions associated with ubiquitination continues to grow with the identification of additional protein substrates targeted by this versatile modification.Like protein phosphorylation, ubiquitination is reversible, and deubiquitination plays an important role in regulating ubiquitin-dependent pathways. Removal of ubiquitin is accomplished by DUBs, 2 a diverse class of nearly 80 enzymes in humans, which cleave ubiquitin from proteins, peptides, or small molecules (6, 7). Although specific substrates have been identified for relatively few DUBs, the importance of this enzyme class is highlighted by recent studies which reveal tight regulation of their activity. Transcriptional regulation, proteinprotein interactions, and post-translational modifications are known to regulate DUB activity (7,8). For example, the DUB USP1 (ubiquitin-specific protease-1) is inactivated by a UV irradiation-induced auto-cleavage event, allowing for accumulat...
Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.
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