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. Such conjugates are highly difficult to prepare homogenously and in workable quantities using the enzymatic machinery. To address this formidable challenge we developed new chemical approaches to covalently attach ubiquitin chains to a protein substrate through its Cys residue. A key aspect of this approach is the installation of acyl hydrazide functionality at the C-terminus of the proximal Ub, which allows, after ubiquitin chain assembly, the introduction of various reactive electrophiles for protein conjugation. Employing α-globin as a model substrate, we demonstrate the facile conjugation to K48-linked ubiquitin chains, bearing up to four ubiquitins, through disulfide and thioether linkages. These bioconjugates were examined for their behavior with the USP2 enzyme, which was found to cleave the ubiquitin chain in a similar manner to unanchored ones. Furthermore, proteasomal degradation study showed that di-ubiquitinated α-globin is rapidly degraded in contrast to the mono-ubiquitinated counterpart, highlighting the importance of the chain lengths on proteasomal degradation. The present work opens unprecedented opportunities in studying the ubiquitin signal by enabling access to site-specifically polyubiquitinated proteins with an increased size and complexity.

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). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.

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. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to ∼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.

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. Here, we demonstrate in a direct manner that Ub is indeed degraded in a "piggyback" mechanism. Also, it has been shown that monomeric Ub can be rapidly degraded when a C-terminal tail of a minimal length is fused to it. The tail, which may represent the substrate or part of it, or a naturally occurring extended form of Ub, probably allows entry of the protein into the 20S catalytic chamber, while Ub serves as an anchor to the 19S complex. Here, we show that shorter-tailed Ubs, such as UBB(+1), bind to the proteasome but because they cannot be efficiently degraded, they inhibit the degradation of other Ub system's substrates such as Myc, p21, Mdm2, and MyoD. The inhibition depends on the ability of the tailed Ubs to be ubiquitinated: their mere binding to the proteasome is not sufficient. Interestingly, the inhibition affects only substrates that must undergo ubiquitination for their degradation: ornithine decarboxylase that is targeted by the proteasome in a Ub-independent manner, is not affected by the short-tailed ubiquitinated Ubs, suggesting it binds to the 19S complex in a site different from that to which ubiquitinated substrates bind.

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). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.