Targeting SUMO conjugates for degradation: The human RING finger RNF4 as a specialized ubiquitin ligase

Ubiquitin and the small ubiquitin related modifier (SUMO) belong to a group of small proteins that can be covalently attached to lysine side chains of other proteins, thereby changing their function, localization, interaction partners or stability. The conjugation reactions are mediated by an enzymatic cascade of specific activating, conjugating and ligating enzymes. A ubiquitin chain of at least four K48-linked ubiquitin molecules target substrate proteins for degradation by the proteasome. Several interconnections exist between the ubiquitin and SUMO system, with the latest discoveries made in yeast by identifying E3 ubiquitin ligases that target SUMO conjugates for ubiquitylation and subsequent degradation by the proteasome. These ubiquitin ligases for SUMO conjugates (ULS) recognize especially high molecular weight SUMO conjugates, probably modified with SUMO chains. In mammals, out of the three conjugatable SUMO paralogs, only SUMO-2/3 are able to form chains. Upon stress induction, the free pool of SUMO-2/3 is rapidly conjugated to cellular target proteins. These conjugates are under proteasomal control, implicating that the ULS pathway is conserved in humans. This work identified the RING finger protein RNF4 as a human ULS protein, confirming previous observations in which RNF4 complemented yeast ULS deletion phenotypes. RNF4 comprises a RING domain which is present in many E3 ligases and a stretch of up to four SUMO interaction motifs (SIMs) that confer binding to SUMO. In order to demonstrate ULS activity for RNF4, an in vitro ubiquitylation assay for SUMO conjugates has been developed. For that purpose, SUMOylated proteins were generated and purified as in vitro substrates from E. coli. RNF4 efficiently in vitro ubiquitylated SUMO modified PML while unmodified PML was not recognized as a substrate. This result is in line with recent studies in cells demonstrating that RNF4 targets PML in a SUMO-dependent manner after arsenic trioxide treatment, a drug which is applied in acute promyelocytic leukemia (APL). By investigating the SUMO binding properties of the RNF4 SIM domain, it became apparent that the interaction was especially enhanced by the presence of SUMO chains of more than two SUMOs. In addition, a SIM type specific recognition was noticed for different SUMO paralogs, which emphasizes the idea that there is also a SUMO paralogs specific regulation. Finally, in an attempt to find other ULS regulated cellular proteins, an RNF4 SIM domain construct was used to isolate poly- or multi-SUMOylated proteins from cells subjected to diverse cell stresses

Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present

The Journal of Immunology Critical Role for the Tapasin-Docking Site of TAP2 in the Functional Integrity of the MHC Class I-Peptide-Loading Complex 1

The transporter associated with Ag processing (TAP) translocates antigenic peptides into the endoplasmic reticulum for binding onto MHC class I (MHC I) molecules. Tapasin organizes a peptide-loading complex (PLC) by recruiting MHC I and accessory chaperones to the N-terminal regions (N domains) of the TAP subunits TAP1 and TAP2. To investigate the function of the tapasin-docking sites of TAP in MHC I processing, we expressed N-terminally truncated variants of TAP1 and TAP2 in combination with wild-type chains, as fusion proteins or as single subunits. Strikingly, TAP variants lacking the N domain in TAP2, but not in TAP1, build PLCs that fail to generate stable MHC I-peptide complexes. This correlates with a substantially reduced recruitment of accessory chaperones into the PLC demonstrating their important role in the quality control of MHC I loading. However, stable surface expression of MHC I can be rescued in post-endoplasmic reticulum compartments by a proprotein convertase-dependent mechanism. The Journal of Immunology, 2005, 175: 5104–5114. The transporter associated with Ag processing (TAP) 3 transports cytosolic peptides, predominantly generated by the proteasome into the endoplasmic reticulum (ER) (1). There, they are loaded onto newly synthesized MHC class I (MHC I) that transiently associates with TAP and accessory chaperones to form the MHC I peptide-loading complex (PLC) (1)

Different types of ISG15 modification of endogenous Ubc13.

<p>HeLa cells were transiently transfected with pCMVb-HA-Ubc13 WT (A) or pCMVb-HA-Ubc13 C87G (B) mutant and other vectors as shown in the figure. 24 h post-transfection the cells were collected and lysed in presence 2-ME. The metal-chelate pull-downs were carried out under denatured conditions with or without 2-ME. Immunoblotting against the S-tag show the levels of ISG15. Equal loading of total protein was verified by anti alpha-tubulin immunoblotting.</p

Amino acid alignment of ISG15 proteins from different vertebrate species with human di-ubiquitin.

<p>The potential functionally important sites are highlighted. The C-terminal site including the double glycine motif for the conjugation to substrates is marked in bold, cysteine residues are indicated in bold and italics and the conserved cysteine residues in hinge region are shaded in grey. Sequence analysis was performed using ClustalW.</p

No evidence for ISG15 modification of MxA, hGBP1 and PML.

<p>(A) HeLa cells were induced with IFN-β for 24 h. The cells were lysed in urea buffer without reducing agent. Cellular lysates were equally aliquoted and 2-ME was added (loading to SDS-PAGE from left to right: 500 mM, 100 mM, 50 mM, 20 mM, 10 mM, 5 mM, 1 mM 2-ME, empty lane, 0 mM 2-ME) and blotted for MxA and hGBP1. (B) HeLa cells were transiently transfected with pCMV2b-Flag-MxA and the components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were induced with IFN-β24 post-induction the cells were collected and lysed without 2-ME. Anti-FLAG immunoprecipitations were performed without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE (C) HeLa cells were transiently transfected with pCMVb-HA-MxA and components of the ISG15 conjugation machinery as indicated in the figure. 24 h post-transfection, the cells were collected and lysed without 2-ME. Anti-HA immunoprecipitations were performed. Eluates were equally split and treated with or without 2-ME before SDS-PAGE. (D) HeLa cells were transiently transfected with either pCMVb-MRGS-His-ISG15 or pCDNA4/TO/N-MRGS-His-SUMO2. 24 h post-transfection, the cells were induced with IFN-β (1,000 units/ml). Purifications of ISG15 or SUMO2 modified proteins were carried out under denaturating conditions without 2-ME. Eluates were equally split and treated with or without 2-ME before SDS-PAGE.</p

OTULIN Restricts Met1-Linked Ubiquitination to Control Innate Immune Signaling

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling

Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO

RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length