Aubiquitin ligase (also called anE3 ubiquitin ligase) is aprotein that recruits an E2ubiquitin-conjugating enzyme that has been loaded withubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate. In simple and more general terms, the ligase enables movement of ubiquitin from a ubiquitin carrier to another protein (the substrate) by some mechanism. Theubiquitin, once it reaches its destination, ends up being attached by anisopeptide bond to alysine residue, which is part of the target protein.[2] E3 ligases interact with both the target protein and the E2 enzyme, and so impart substrate specificity to the E2. Commonly, E3s polyubiquitinate their substrate with Lys48-linked chains of ubiquitin, targeting the substrate for destruction by theproteasome. However, many other types of linkages are possible and alter a protein's activity, interactions, or localization. Ubiquitination by E3 ligases regulates diverse areas such as cell trafficking, DNA repair, and signaling and is of profound importance in cell biology. E3 ligases are also key players in cell cycle control, mediating the degradation ofcyclins, as well ascyclin dependent kinase inhibitor proteins.[3] The human genome encodes over 600 putative E3 ligases, allowing for tremendous diversity in substrates.[4] Certain E3 ligases have been utilized in targeted protein degradation applications.[5]
The ubiquitylation reaction proceeds in three or four steps depending on the mechanism of action of the E3 ubiquitin ligase. In the conserved first step, an E1cysteine residue attacks the ATP-activated C-terminal glycine on ubiquitin, resulting in athioester Ub-S-E1 complex. The energy from ATP and diphosphate hydrolysis drives the formation of this reactive thioester, and subsequent steps are thermoneutral. Next, a transthiolation reaction occurs, in which an E2 cysteine residue attacks and replaces the E1.HECT domain type E3 ligases will have one more transthiolation reaction to transfer the ubiquitin molecule onto the E3, whereas the much more commonRING finger domain type ligases transfer ubiquitin directly from E2 to the substrate.[6] The final step in the first ubiquitylation event is an attack from the target protein lysine amine group, which will remove the cysteine, and form a stable isopeptide bond.[7] One notable exception to this isp21 protein, which appears to be ubiquitylated using its N-terminal amine, thus forming a peptide bond with ubiquitin.[8]
Humans have an estimated 500-1000 E3 ligases, which impart substrate specificity onto the E1 and E2.[9] The E3 ligases are classified into four families: HECT, RING-finger, U-box, and PHD-finger.[9] The RING-finger E3 ligases are the largest family and contain ligases such as theanaphase-promoting complex (APC) and theSCF complex (Skp1-Cullin-F-box protein complex). SCF complexes consist of four proteins: Rbx1, Cul1, Skp1, which are invariant among SCF complexes, and an F-box protein, which varies. Around 70 human F-box proteins have been identified.[10] F-box proteins contain an F-box, which binds the rest of the SCF complex, and a substrate binding domain, which gives the E3 its substrate specificity.[9]
Ubiquitin with lysine residues (red), N-terminal methionine (blue), and C-terminal glycine (yellow).[11]
Ubiquitin signaling relies on the diversity of ubiquitin tags for the specificity of its message. A protein can be tagged with a single ubiquitin molecule (monoubiquitylation), or variety of different chains of ubiquitin molecules (polyubiquitylation).[12] E3 ubiquitin ligases catalyze polyubiquitination events much in the same way as the single ubiquitylation mechanism, using instead a lysine residue from a ubiquitin molecule currently attached to substrate protein to attack the C-terminus of a new ubiquitin molecule.[7][12] For example, a common 4-ubiquitin tag, linked through the lysine at position 48 (K48) recruits the tagged protein to the proteasome, and subsequent degradation.[12] However, all seven of the ubiquitin lysine residues (K6, K11, K27, K29, K33, K48, and K63), as well as the N-terminal methionine are used in chains in vivo.[12]
Monoubiquitination has been linked to membrane proteinendocytosis pathways. For example, phosphorylation of the Tyrosine at position 1045 in theEpidermal Growth Factor Receptor (EGFR) can recruit the RING type E3 ligase c-Cbl, via anSH2 domain. C-Cbl monoubiquitylates EGFR, signaling for its internalization and trafficking to the lysosome.[13]
Monoubiquitination also can regulate cytosolic protein localization. For example, the E3 ligaseMDM2 ubiquitylatesp53 either for degradation (K48 polyubiquitin chain), or for nuclear export (monoubiquitylation). These events occur in a concentration dependent fashion, suggesting that modulating E3 ligase concentration is a cellular regulatory strategy for controlling protein homeostasis and localization.[14]
Ubiquitin ligases are the final, and potentially the most important determinant ofsubstrate specificity inubiquitination ofproteins.[15] The ligases must simultaneously distinguish their protein substrate from thousands of other proteins in thecell, and from other (ubiquitination-inactive) forms of the same protein. This can be achieved by different mechanisms, most of which involve recognition ofdegrons: specific shortamino acid sequences or chemical motifs on the substrate.[16]
Proteolytic cleavage can lead to exposure of residues at theN-terminus of a protein. According to theN-end rule, different N-terminal amino acids (or N-degrons) are recognized to a different extent by their appropriate ubiquitin ligase (N-recognin), influencing thehalf-life of the protein.[17] For instance, positively charged (Arg,Lys,His) and bulkyhydrophobic amino acids (Phe,Trp,Tyr,Leu,Ile) are recognized preferentially and thus considered destabilizingdegrons since they allow faster degradation of their proteins.[18]
A phosphorylated degron (green) is stabilized by hydrogen bonding (yellow) between oxygen atoms of its phosphate (red) and side chains of the SCFFBW7ubiquitin ligase (blue). The relevant part of the ubiquitin ligase is shown in gray. PDB entry 2ovr[19]
A degron can be converted into its active form by apost-translational modification[20] such asphosphorylation of atyrosine,serine orthreonine residue.[21] In this case, the ubiquitin ligase exclusively recognizes the phosphorylated version of the substrate due to stabilization within thebinding site. For example,FBW7, theF-box substrate recognition unit of anSCFFBW7ubiquitin ligase, stabilizes a phosphorylated substrate byhydrogen binding itsarginine residues to the phosphate, as shown in the figure to the right. In absence of thephosphate, residues of FBW7 repel the substrate.[19]
The presence ofoxygen or other smallmolecules can influence degron recognition.[19] Thevon Hippel-Lindau (VHL) protein (substrate recognition part of a specific E3 ligase), for instance, recognizes thehypoxia-inducible factor alpha (HIF-α) only under normal oxygen conditions, when itsproline ishydroxylated. Underhypoxia, on the other hand, HIF-a is not hydroxylated, evadesubiquitination and thus operates in the cell at higher concentrations which can initiatetranscriptional response to hypoxia.[22] Another example of small molecule control of protein degradation isphytohormoneauxin in plants.[23] Auxin binds to TIR1 (the substrate recognition domain ofSCFTIR1ubiquitin ligase) increasing the affinity of TIR1 for its substrates (transcriptionalrepressors: Aux/IAA), and promoting their degradation.
In addition to recognizing amino acids, ubiquitin ligases can also detect unusual features on substrates that serve as signals for their destruction.[15] For example, San1 (Sir antagonist 1), anuclear protein quality control inyeast, has a disordered substratebinding domain, which allows it to bind to hydrophobic domains ofmisfolded proteins.[15] Misfolded or excess unassembledglycoproteins of theERAD pathway, on the other hand, are recognized byFbs1 and Fbs2, mammalianF-box proteins of E3 ligasesSCFFbs1and SCFFbs2.[24] These recognition domains have small hydrophobic pockets allowing them to bind high-mannose containingglycans.
In addition to lineardegrons, the E3 ligase can in some cases also recognizestructural motifs on the substrate.[15] In this case, the 3D motif can allow the substrate to directly relate itsbiochemical function toubiquitination. This relation can be demonstrated withTRF1 protein (regulator of humantelomere length), which is recognized by its corresponding E3 ligase (FBXO4) via anintermolecularbeta sheet interaction. TRF1 cannot be ubiquinated while telomere bound, likely because the same TRF1 domain that binds to its E3 ligase also binds to telomeres.[15]
E3 ubiquitin ligases regulate homeostasis, cell cycle, and DNA repair pathways, and as a result, a number of these proteins are involved in a variety of cancers, including famously MDM2,BRCA1, andVon Hippel-Lindau tumor suppressor.[25] For example, a mutation of MDM2 has been found instomach cancer,[26]renal cell carcinoma,[27] andliver cancer[28] (amongst others) to deregulate MDM2 concentrations by increasing its promoter's affinity for theSp1 transcription factor, causing increased transcription of MDM2 mRNA.[26] Several proteomics-based experimental techniques are available for identifying E3 ubiquitin ligase-substrate pairs,[29] such as proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, and tandem ubiquitin-binding entities (TUBEs).
ARING (ReallyInterestingNewGene) domain binds the E2 conjugase and might be found to mediate enzymatic activity in the E2-E3 complex[30]
An F-box domain (as in the SCF complex) binds the ubiquitinated substrate. (e.g., Cdc 4, which binds the target proteinSic1; Grr1, which binds Cln).[31]
AHECT domain, which is involved in the transfer of ubiquitin from the E2 to the substrate.
In 2001, work from the labs ofCraig Crews andRaymond Deshaies described the development ofproteolysis-targeting chimeras (PROTACs).[32] Using a small molecule to recruit an E3 ubiquitin ligase to a target protein, this work demonstrated that induced proximity could be used to effect the ubiquitination and proteasomal degradation of a target protein. PROTACs have been frequently applied using the E3 ubiquitin ligases CRBN[33][34] and VHL[35][36] to degrade various targets of biological and therapeutic relevance. Multiple groups have sought out additional E3 ligases to co-opt for targeted protein degradation such as FBXO22[37][38][39] and KLHDC2.[40]
While PROTACs generally are heterobifunctional compounds linking an E3 ligase binder to a target protein binder,molecular glues also exist that induceprotein-protein interactions with E3 ligases, leading to degradation of various substrate proteins. Molecular glues often have been discovered through serendipity,[41][42][43] though various methodologies have been explored to expedite the discovery of molecular glues.[44][45][46][47][48][49]
Biologic modalities for targeted protein degradation have also been explored by fusing E3 ligases to target recognition domains such asnanobodies. These modalities are sometimes referred to as bioPROTACs.[50][51] While bioPROTACs are advantageous for targeting proteins lacking small molecule ligands, challenges in delivery, pharmacokinetics, and immunogenicity have so far precluded clinical development.[52] Studies exploring different delivery mechanisms have sought to address these shortcomings.[53] In another variant of this idea, bispecific antibodies to recruit membrane-bound E3 ligases to cell surface proteins (AbTACs) have also been developed.[54]
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