br Author contribution br Acknowledgements We want to thank
Acknowledgements We want to thank Dr. Manuel Rodriguez, Dr. Ch. Blattner, Dr. Jorge Moscat, Dr. Erwin Knecht and Dr. Peter Tontonoz for materials. This work has been supported by grants from the Spanish Ministry of Education and ScienceSAF2011-27442 and a grant from Generalitat Valenciana (PrometeoII/2014/029).
Introduction Protein ubiquitination initiates with the ATP-dependent activation of ubiquitin (Ub) by ubiquitin-activating enzyme (E1), where a thioester linkage is formed between the C-terminus of ubiquitin and the active site cysteine (Cys) of E1 (Fig. 1). Ubiquitin is then transthiolated to the active site of one of ~40 different (in mammals) ubiquitin-conjugating Meptazinol HCl (E2), generating an E2~Ub thioester. Specificity of ubiquitin modification is achieved largely through ubiquitin protein ligases (E3), which interact with both E2~Ub and the substrate to which ubiquitin is to be transferred. Ubiquitination generally occurs on primary amines as a consequence of nucleophilic attack on the E2~Ub linkage, resulting in stable isopeptide (or peptide) linkages with the C-terminus of ubiquitin. Ubiquitin can be transferred either to a lysine (Lys), or (less frequently) to the N-terminus, of either a substrate or another ubiquitin molecule to generate multi- or poly-ubiquitin chains. To a large extent, the nature of these ubiquitin linkages specifies the fate and function of the modified protein (see Fig. 1 and legend for more detail). E3s function by one of two general mechanisms. They serve either as catalytic intermediates in ubiquitination, akin to E1 and E2, or they mediate the transfer of ubiquitin directly from E2~Ub to substrate. The former mechanism is used by Homologous to E6-AP Carboxy Terminus (HECT)-type E3s (reviewed in this issue by Martin et al). The latter mechanism is a characteristic of Really Interesting New Gene (RING)-type E3s. RINGs coordinate two Zn ions in a cross-braced arrangement (Fig. 2 and described in detail below) to create a platform for binding of E2s. The RING-like U-box family of E3s adopts a similar structure to bind E2s but without employing Zn coordination. Together, RING (also known as RING finger, RING motif, or RING domain) and RING-like E3s (plant homeodomain/leukemia-associated protein (PHD/LAP) and U-box), collectively referred to as ‘RING-type’ in this review, constitute the large majority of the over 600 E3s in mammalian cells. Any consideration of RING-type E3s must include their partners, the E2s. E2s have a core conserved ubiquitin-conjugating (UBC) domain that contains a conserved catalytic Cys (Fig. 2). Some E2s have insertions within the UBC domain or N- or C-terminal extensions that confer specific functions. For HECT E3s, ubiquitin chain linkage specification lies largely with the catalytic HECT domain, but the situation is more complicated for RING-type E3s and their E2s. Some E2s are dedicated to specific ubiquitin linkages; other E2s are intrinsically more promiscuous with respect to the linkages they generate. Thus, a given RING-type E3 can generate different ubiquitin linkages depending on the E2 with which it is paired. Structural and functional properties of E2s have been reviewed in detail elsewhere . Mutation of RING-type E3s, or modulation of their activity in other ways, is often associated with human disease. BRCA1, an E3 that plays critical roles in DNA repair, is mutated in familial breast and ovarian cancers . Mutations in components of the FANC ubiquitin ligase, also involved in DNA repair, result in the multisystemic Fanconi anemia syndrome , which includes severe developmental defects and, in children who survive, there is a marked increased risk of tumor development. Mdm2 (or Hdm2 in humans) was first characterized as a genetic amplification in mice associated with malignancy . Indeed, increased activity of this E3 towards the tumor suppressor p53, either through increased Mdm2 expression or loss of a negative modulator of Mdm2 activity, is associated with human cancers, particularly those 50% that retain wild type p53 . The F-box protein FBXO11, the substrate recognition component of the multi-subunit SCFFBXO11 E3 (see Section 3 below), functions as a tumor suppressor by targeting BCL6, a transcription factor involved in B-cell differentiation and activation, for degradation . FBXO11 is mutated or deleted in diffuse large B-cell lymphomas . Mutations in VHL, the substrate recognition component of the CRL2VHL E3, lead to the malignant von Hippel–Lindau syndrome, which presumably arises because of dysregulation of HIF1-α and/or HIF2-α . Mutations in the RING–IBR (in between RING)–RING E3 Parkin are associated with autosomal recessive juvenile Parkinsonism (AR-JP) . Additionally, a number of viruses, for example, herpes simplex virus type 1 (HSV-1), encode RING-type E3s as virulence factors , . In the case of HIV, the virus encodes an adaptor protein, Vpu, that redirects SCFβTrCP to downregulate CD4 . The importance of RING-type E3s to human health and disease has contributed to their becoming an intensively-studied family of proteins. This review will provide an overview of their regulated function and structure and recent advances in understanding how they mediate ubiquitination by E2s.