Search

Nedd8 is a ubiquitin-like molecule that is highly conserved in eukaryotes. Similar to ubiquitin, Nedd8 attaches to target proteins through an enzymatic cascade composed of Nedd8-specific E1 (activating)- and E2 (conjugating)-enzymes. The E1 for Nedd8 is a heterodimer of APP-BP1 and Uba3, while the E2 is Ubc12. The most well-characterized targets of Nedd8 are proteins of the Cullin family, a core component of SCF (Skp1/Cullin1/F-box proteins) and/or SCF-like ubiquitin ligase complexes. The Nedd8 modification of Cullin (Cul) family proteins is evolutionarily conserved, and genetic analyses in various organisms suggest a positive role of the NEDD8 for the function of Cul family proteins. Further biochemical analysis reveals that NEDD8 modification augments the ubiquitin ligase activity of Cullin-based complexes through the recruitment of ubiquitin-charged E2 to the complex. This chapter describes methods for the purification of NEDD8 conjugation enzymes and in vitro Nedd8 conjugation.

Publisher Summary Park2 (autosomal recessive-juvenile parkinsonism (AR-JP)) presents young-onset parkinsonism, consisting of gait disturbance, rest tremor, cogwheel rigidity, and bradykinesia. Clinical features are essentially similar to those of late-onset sporadic Parkinson's disease. They respond to levodopa well. Progression is slow. Pathologic features include extensive nigral and locus coeruleus degeneration and gliosis without Lewy body formation. The disease gene is identified and named parkin, which is located on the long arm of chromosome 6 at 6q25-27.2. Varieties of deletion mutations and point mutations of parkin are found in patients with Park2. Compound heterozygotes are also found. Parkin protein functions as a ubiquitin ligase and a number of candidate substrates for Parkin are reported including CDCrel 1, α-synuclein 22, Pael receptor, synphilin-1, and CDCrel 2A. Accumulation of one or more of the candidate substrates appears to be the cause of nigral degeneration. Park2 is considered to represent the most common form of familial Parkinson's disease.