Nonenzymatic polyubiquitination of expressed proteins.

Ubiquitination is one of the most ubiquitous posttranslational modifications in eukaryotes and is involved in various cellular events such as proteasomal degradation and DNA repair. The overwhelming majority of studies aiming to understand ubiquitination and deubiquitination have employed unanchored ubiquitin chains and mono-ubiquitinated proteins. To shed light on these processes at the molecular level, it is crucial to have facile access to ubiquitin chains linked to protein substrates.

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology.

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn).

The size of the proteasomal substrate determines whether its degradation will be mediated by mono- or polyubiquitylation.

A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal.

Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation.

The mechanisms that regulate the ubiquitin (Ub)-proteasome system's own components, although critically important, are largely unknown. Ub, a principal component of the system, must be maintained at adequate levels to support cellular homeostasis under basal and stressed conditions. It was suggested that Ub is degraded as part of the polyubiquitin chain along with its substrate.

SIAH-1 interacts with the Kaposi's sarcoma-associated herpesvirus-encoded ORF45 protein and promotes its ubiquitylation and proteasomal degradation.

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH).