Breaking the limits of artificial ubiquitination.

Neurodegenerative diseases such as Parkinson, Alzheimer’s, and Huntington are proteopathic disorders characterized by the accumulation of intracellular insoluble aggregates of misfolded proteins and are one of the major medical concerns of our modern society. Particularly, Parkinson disease is a synucleinopathy caused by the cytosolic aggregation of α-Synuclein into Lewy bodies (1). Although its biological function remains obscure, numerous lines of evidence clearly point at α-Synuclein oligomers or aggregates as the causative agent of Parkinson disease (1). However, despite intense research, the detailed etiology of Parkinson disease remains elusive. Apart from missense point mutations in the α-Synuclein gene (which account for familial cases of early onset Parkinson disease), increased intracellular expression levels and posttranslational modifications of αSynuclein have been implicated in the formation of Lewy bodies. Currently, known modifications of α-Synuclein include proteolytic truncation, oxidation, nitration, phosphorylation, and ubiquitination (1). The key to understanding proteopathic diseases such as Parkinson disease rests on elucidating the role of these modifications and how they influence the properties of the nascent αSynuclein polypeptide. In their current research article, Haj-Yahya et al. report on an important milestone in the synthesis of selectively diand tetra-ubiquitinated (K48) α-Synuclein, which can be used to study the biophysical and biochemical consequences of α-Synuclein ubiquitination (2). In addition to offering valuable insights into molecular biology of synucleinopathies, their findings also open up new horizons in the research on ubiquitination—a crucial posttranslational protein modification involved in quintessential aspects of cell biology (3). Ubiquitin is a small protein of 76 amino acids and is highly conserved among all eukaryotic organisms. Ubiquitin functions as a posttranslational protein modifier that is covalently attached to lysine residues on the target substrates by its C-terminal glycine (4). Protein targets become ubiquitinated by an enzymatic transfer reaction, which involves three types of enzymes: E1, the ubiquitin-activating enzyme; E2, the ubiquitin-conjugating enzyme; and an E3 ubiquitin ligase. The concerted action of this enzymatic triad is opposed by deubiquitinating enzymes, which remove ubiquitin moieties from modified target proteins, thus contributing to highly dynamic changes in the ubiquitination status of the substrate proteins. Interestingly, the human genome encodes >600 E3 ubiquitin ligases and ∼100 deubiquitinases, clearly illustrating the complexity of the intracellular machinery involved in producing ubiquitin signals (5, 6). Importantly, ubiquitin itself contains seven lysine residues enabling assembly of seven homotypic types of polymeric ubiquitin chains. Notably, assembly of ubiquitin chains can also be achieved by head-to-tail fusion of ubiquitin moieties, in that C-terminal glycine of ubiquitin is conjugated to N-terminal amino group of the next ubiquitin molecule, giving rise to linear ubiquitin chains (7). The structural, biophysical, and signaling properties of these distinct types of ubiquitin chains are determined by the specific type of linkage between single ubiquitin molecules within the polymeric chain, which can be recognized by numerous ubiquitin binding domains of a diverse range of effector proteins (7). Despite the remarkable progress reached over the past decades of studying ubiquitination, further advances are severely hampered by our inability to obtain workable amounts α-synuclein polyubiqui n chain (K48)