The K48-K63 Branched Ubiquitin Chain Regulates NF-κB Signaling

Abstract

Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1β, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control

“…Such experiments suggested that M1/K63-linked chains control NFκB transcription factor activation (Emmerich et al, 2016; Emmerich et al, 2013; Wertz et al, 2015), while K11/K48-branched chains signal the degradation of cell cycle regulators during mitosis (Meyer and Rape, 2014). Similar in vitro or protein engineering evidence exists for K48/K63-branched, K29/K48-linked or more complex heterotypic chains (Kim et al, 2007; Koegl et al, 1999; Kristariyanto et al, 2015; Liu et al, 2017; Ohtake et al, 2016). These findings implied that cells use heterotypic chains for signal transduction, yet without a method to monitor endogenous polymers in vivo , the abundance, function, and importance of mixed or branched ubiquitin chains remain very poorly understood.…”

Lys63-polyubiquitination by the E3 ligase casitas B-lineage lymphoma-b (Cbl-b) modulates peripheral regulatory T cell tolerance in patients with systemic lupus erythematosus

“…As prior studies have illustrated numerous roles of Lys63-linked (K63) polyubiquitylation in immune responses [39][40][41][42][43][44][45], as well as its relationship with Cbl-b [27], we determined if the deficiency in this E3 ubiquitin ligase could be associated with an abnormal polyubiquitin profile of T regs from SLE patients in a K63-dependent manner. According to our hypothesis, we observed a differential polyubiquitination profile distinguished by a decreased expression of K63 substrates in lupus T regs , but not in effector T cells.…”

“…Lysine-63-specific protein ubiquitination has been implicated in the regulation of signal transduction in immune receptors [39][40][41][42][43][44][45], which is mediated by the E2 ubiquitinconjugating enzyme Ubc13, as shown by multiple studies, and directs conjugated proteins to lysosomes [44]. As Ubc13 inducible knock-out mice specific for T regs display a reminiscent pathological phenotype of the T reg -deficient mice [46], we hypothesized that Cbl-b deficiency would modify the ubiquitin profile of T regs from SLE patients in a K63-dependent manner, which could contribute to resistance to suppression.…”

Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome

“…Ubiquitin itself has 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63) along with its N-terminal methionine, resulting in the potential for multiple types of ubiquitin chains. Some of these linkages can be monomeric, polymeric, linked in tandem, branched, or even unanchored, with homogeneous or heterogeneous types of linkages [22,27,28]. In contrast to phosphorylation, multiple types of ubiquitin chains can be constructed and added to target proteins, which dramatically increases the amount of signaling information that can be encoded by ubiquitination.…”

“…It is important to reiterate that different ubiquitin linkages lead to different outcomes for target proteins. K48- and K63-linked chains are the most abundant linkages in cells and are also the most extensively characterized [22,29]. K48-linked polyubiquitin chains generally direct target proteins to the proteasome for degradation [22].…”