Aspects of how Burkholderia escape the host\'s intrinsic immune response to replicate in the cell cytosol remain enigmatic. Here, we show that Burkholderia has evolved two mechanisms to block the activity of Ring finger protein 213 (RNF213)-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide (LPS), thereby preventing the initiation of antibacterial autophagy. First, Burkholderia\'s polysaccharide capsule blocks RNF213 association with bacteria and second, the Burkholderia deubiquitylase (DUB), TssM, directly reverses the activity of RNF213 through a previously unrecognized esterase activity. Structural analysis provides insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from its isopeptidase function. Furthermore, a putative TssM homolog also displays esterase activity and removes ubiquitin from LPS, establishing this as a virulence mechanism. Of note, we also find that additional immune-evasion mechanisms exist, revealing that overcoming this arm of the host\'s immune response is critical to the pathogen.

The post-translational modification of proteins with ubiquitin plays a central role in nearly all aspects of eukaryotic biology. Historically, studies have focused on the conjugation of ubiquitin to lysine residues in substrates, but it is now clear that ubiquitylation can also occur on cysteine, serine, and threonine residues, as well as on the N-terminal amino group of proteins. Paradigm-shifting reports of non-proteinaceous substrates have further extended the reach of ubiquitylation beyond the proteome to include intracellular lipids and sugars. Additionally, results from bacteria have revealed novel ways to ubiquitylate (and deubiquitylate) substrates without the need for any of the enzymatic components of the canonical ubiquitylation cascade. Focusing mainly upon recent findings, this review aims to outline the current understanding of non-lysine ubiquitylation and speculate upon the molecular mechanisms and physiological importance of this non-canonical modification.

Post-translational protein modifications initiate, regulate, propagate and terminate a wide variety of processes in cells, and in particular, ubiquitylation targets substrate proteins for degradation, subcellular translocation, cell signaling and multiple other cellular events. Modification of substrate proteins is widely observed to occur via covalent linkages of ubiquitin to the amine groups of lysine side-chains. However, in recent years several new modes of ubiquitin chain attachment have emerged. For instance, covalent modification of non-lysine sites in substrate proteins is theoretically possible according to basic chemical principles underlying the ubiquitylation process, and evidence is building that sites such as the N-terminal amine group of a protein, the hydroxyl group of serine and threonine residues and even the thiol groups of cysteine residues are all employed as sites of ubiquitylation. However, the potential importance of this \"non-canonical ubiquitylation\" of substrate proteins on sites other than lysine residues has been largely overlooked. This review aims to highlight the unusual features of the process of non-canonical ubiquitylation and the consequences of these events on the activity and fate of a protein.