Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 ...conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which ...induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
RING E3 ligase-catalyzed formation of K63-linked ubiquitin chains by the Ube2V2-Ubc13 E2 complex is required in many important biological processes. Here we report the structure of the RING-domain ...dimer of rat RNF4 in complex with a human Ubc13∼Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with K63 in a position favorable for attack on the linkage between Ubc13 and the donor (second) ubiquitin held in the active 'folded back' conformation by the RING domain of RNF4. We verified the interfaces identified in the structure by in vitro ubiquitination assays of site-directed mutants. To our knowledge, this represents the first view of synthesis of K63-linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase-mediated catalysis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural ...disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.
The small ubiquitin‐like modifier (SUMO) can undergo self‐modification to form polymeric chains that have been implicated in cellular processes such as meiosis, genome maintenance and stress ...response. Investigations into the biological role of polymeric chains have been hampered by the absence of a protocol for the purification of proteins linked to SUMO chains. In this paper, we describe a rapid affinity purification procedure for the isolation of endogenous polySUMO‐modified species that generates highly purified material suitable for individual protein studies and proteomic analysis. We use this approach to identify more than 300 putative polySUMO conjugates from cultured eukaryotic cells.
The authors describe a rapid affinity purification procedure for the isolation of endogenous polySUMO modified proteins, which provides highly purified material suitable for individual protein studies as well as proteomic analysis.
Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight ...into the mechanism of RING-mediated ubiquitylation, we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model, the 'Ile44 hydrophobic patch' of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING, and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ...ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.
The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein ...turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13-UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.
Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase ...containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates.
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•Characterization of the mechanism of activation of the ubiquitin E3 ligase RNF4•First indication of E3 ligase activation by SUMO chains
RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. Rojas-Fernandez et al. show that in the presence of SUMO chains RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation.
Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. ...Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.
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