Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C ter- minus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibi- tory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruit- ment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein–protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways. Keywords Ubiquitination · HECT E3 ligase · Substrate recruitment · Intramolecular interaction · Activity regulation · Oligomerization · Post-translational modification Introduction regulation and this makes them attractive targets for thera- peutic intervention [1–3]. Ubiquitination is a post-translational modification that is E3 ligases are commonly grouped into three classes: important for regulating protein function and degradation. really interesting new genes (RINGs), homologous to E6AP The ubiquitination cascade comprises the sequential actions C terminus (HECTs), and RING-between-RINGs (RBRs). of the ubiquitin-activating enzyme (E1), ubiquitin-conju- While the three classes of E3 ligases all catalyze covalent gating enzymes (E2s), and ubiquitin ligases (E3s) (Fig. 1). attachment of ubiquitin to usually a Lys residue in the target Within the ubiquitin cascade, the E3 ligases primarily deter- protein, they differ in structure and mechanisms (reviewed mine specificity regarding selection of target proteins and in ). A notable distinction in the mechanism between ubiquitination sites. E3s are often focal points of cellular the classes is that RING E3s catalyze a direct transfer of ubiquitin from the E2 to the target protein, whereas transfer of ubiquitin by HECT E3s (and RBR ligases) involves and intermediate step where the ubiquitin is first transferred from * Ben Distel the E2 to an active-site cysteine residue on the HECT E3 firstname.lastname@example.org ligase before it is conjugated to the target protein. Target proteins can be modified by a single ubiquitin moiety on Department of Medical Biochemistry, Academic one or multiple sites, giving rise to mono- and multi-mono- Medical Center, University of Amsterdam, Amsterdam, The Netherlands ubiquitinated proteins, respectively. In addition, a wide vari- ety of polyubiquitin chains can be formed on target proteins, Department of Neuroscience, Erasmus Medical Center, Wijtemaweg 80, 3015 CN Rotterdam, The Netherlands Vol.:(0123456789) 1 3 3122 J. Sluimer, B. Distel Fig. 1 The ubiquitination cascade. The initial step involves the ATP- cally bound to an active-site cysteine of the HECT domain before it dependent transfer of ubiquitin to an active-site cysteine residue on is attached to the target protein. Target proteins can be modified by the E1 ubiquitin-activating enzyme. In the next step, the ubiquitin mono-, multi-mono-, or polyubiquitin. As ubiquitin has seven internal is transferred from the E1 to the E2 ubiquitin-conjugating enzyme. lysine residues and an N-terminal amino group, all of which can be Once the E2 is charged with ubiquitin, it can associate with the E3 to ubiquitinated, a wide variety of polyubiquitin chains can be formed prepare the ubiquitin transfer to the target protein. A RING-E3 will (not shown). Depending on the type of (poly)ubiquitin modification, mediate a direct transfer of ubiquitin from the E2 to the target protein. either the function/localization of the target protein is changed (“reg- The substrate ubiquitination by HECT E3s (and RBR ligases, not ulation”) or the target protein is sent for degradation by the 26S pro- shown) involves an additional step where the ubiquitin is first chemi- teasome (“degradation”) in which the ubiquitin moieties can be linked through either syndrome which is caused by the loss of maternally inher- one of the seven internal Lys residues (Lys6, Lys11, Lys27, ited UBE3A [9, 10]. E6AP was discovered through its inter- Lys29, Lys33, Lys48, or Lys63) in ubiquitin or through its action with the human papillomavirus protein (HPV) E6, N-terminal amino group. The consequences for the modified which hijacks the E3 ligase to ubiquitinate the p53 tumor target protein are determined by the type of (poly)ubiqui- suppressor as well as several other cellular proteins resulting tin modification it received. Ubiquitination can result in the in their degradation [11, 12]. In a non-infected cell p53 is change of function, localization or activity of the modified not a target of E6AP, but the E6 protein alters the substrate protein, or control its degradation via the 26S proteasome specificity of the E3 ligase to target and ubiquitinate p53 (reviewed in ). . The interaction between the E6 protein and E6AP is one The identification of the E6AP protein transcribed from example of how a HECT E3 ligase is activated and altered the ubiquitin-protein ligase E3A (UBE3A) gene led to the to ubiquitinate a specific substrate through binding of an discovery of the HECT-type E3 ligase family [6–8]. HECT adaptor protein. E3s are directly implicated in cancer, hypertension, neurode- The interaction with adaptor proteins, such as the inter- generative disorders, and other diseases, such as Angelman action of E6 with E6AP, can not only change the substrate 1 3 Regulating the human HECT E3 ligases 3123 specificity of HECT E3s but also alter the structure of HECT E3 ligases the ubiquitin polymers they conjugate to the substrates. Post-translational modifications (PTMs) are also preva - HECT E3 ligases can be distinguished from other classes lent in the alteration of HECT E3 ligase functionality such of ubiquitin E3 ligases in that they have an active-site as phosphorylation and the attachment of ubiquitin-like cysteine that forms an intermediate thioester bond with modifiers (UBLs) [13– 15]. Various mechanisms other than ubiquitin before the ubiquitin is linked to its substrate PTMs and interactions with adaptor proteins also regulate [8, 18]. RING E3s do not have this catalytic activity, but HECT E3 ligases such as intramolecular interactions, oli- rather act as allosteric activators of E2s that mediate the gomerization, and recruitment of the E2 (Fig. 2). transfer of ubiquitin from E2 to substrate directly . This review aims to give a comprehensive overview of This direct transfer of ubiquitin from the E2 means that the different mechanisms through which HECT E3 ligase the linkage type of the ubiquitin chains catalyzed by RING activities and substrate specificities are regulated. The E3s is generally determined by the specific E2 conjugat - structural aspects of these regulatory mechanisms have ing enzyme . In contrast, HECT E3 ligases form an been very recently surveyed , while the pathophysi- E3 ~ Ub intermediate prior to the transfer of ubiquitin to ological aspects of HECT E3 ligases have been addressed the substrate, allowing them to override any linkage-type by Scheffner and Kumar . In addition to providing a preferences that an E2 conjugating enzyme may have . clearer perspective of the regulation of HECT E3 ligases, Consistent with this, some HECT E3s appear to have a this overview could show us which aspects of research on general preference for catalyzing chains of specific linkage HECT E3s have made good progress and which areas of types even when they cooperate with different E2s: E6AP research have lagged behind. Together, these insights may predominantly assembles Lys48-linked chains [21–23]; lead to suggestions for future research and pave the way for Rsp5 and NEDD4.1 assemble preferentially Lys63-linked new therapeutic strategies for many diseases. chains [21–23]; UBE3C promotes formation of Lys29- and Lys48-linked chains ; and recently it was shown that WWP1 assembles ubiquitin chains containing Lys63, Lys48, and Lys11 linkages . For most other HECT E3s their linkage-type preferences, if any, remain to be discovered. Along with ubiquitin, HECT E3s are found in all eukaryotic organisms. HECT domain-like E3 proteins have also been found in pathogenic bacteria . These bacteria presumably exploit the ubiquitin system of their host cells by injecting them with the respective HECT domain-like E3s . Another characteristic of some HECT E3 ligases is that they are capable of catalyzing UBL proteins to their substrates such as NEDD8 (neural precursor cell expressed developmentally down-regulated protein 8)  and ISG15 (ubiquitin-like modifier IFN- stimulated gene 15) [27–29]. The HECT domain The HECT domain is an approximately 40 kDa domain positioned at the C-terminal of the E3 ligases that consists of two flexibly tethered lobes (the N- and C-lobes). Across the HECT family, there is a 16–92% amino-acid identity for the domain . The larger N-lobe (approximately Fig. 2 Modes of regulating HECT E3 ligase function. Mechanisms 250 amino acids) contains the docking surface for the E2 that can regulate or modulate HECT E3 ligase function are: (1) recruitment of substrate and activity modulation by adaptor proteins/ . The short flexible hinge connects the N-lobe to the co-activators, (2) recruitment of E2, (3) intramolecular interaction shorter C-lobe, which contains the active-site cysteine. between an N-terminal domain and the HECT domain, (4) intermo- Non-covalent interactions between the E2 and the N- lecular interaction/oligomerization, (5) post-translational modifica- and C-lobes influence the conformation of the HECT-E2 tion, and (6) ubiquitin binding to the “exosite” on the HECT domain. For details see text 1 3 3124 J. Sluimer, B. Distel complex depending on the ubiquitin-loading status of the 1(RCC)-like domains (RLD). Yeast has five HECT E3 E2. A crystal structure of the E6AP HECT domain with ligases: Rsp5, Ufd4, Hul4, Hul5, and Tom1. Rsp5 is a an unloaded E2 shows a large distance between the E2 member of the NEDD4 family, whereas the other four yeast and HECT domain cysteines . A subsequent struc- HECT E3s do not belong to any family. Rsp5 is also the only ture of the NEDD4.2 HECT with an ubiquitin-loaded E2 HECT E3 that is essential for the viability of the yeast. Each revealed a large change in E2–E3 topology, bringing the of the members of the “other” human HECT E3s lack WW cysteines of both proteins in close proximity  (Fig. 3). or RLD domains and have a distinct variety of N-terminal These structural rearrangements, which are required for domains (Fig. 4). catalysis, are dependent on the flexibility of the linker that connects the N- and C-lobes of the HECT domain . NEDD4 subfamily Properties of the E2 docking surface on the HECT domain in combination with the E2-conjugating enzyme involved The NEDD4 subfamily is the largest and best character- may determine the efficiency at which ubiquitin chains are ized family of the HECT E3s. The N-terminal C2 domain 2+ elongated. This is presumed because polyubiquitin chain is defined as a Ca and phospholipid binder . Consist- synthesis requires multiple E2–E3 binding events due to ent with observed NEDD4 ligase functions, C2 domains an overlapping binding domain on the E2 for E1 and E3 are known for targeting their proteins to phospholipid . membranes . The C2 domain can also bind to substrate proteins to target them for ubiquitination [37, 38]. In some NEDD4 E3s, the C2 domain is involved in regulating the HECT E3 families activity of the HECT domain, as it is capable of binding to the HECT domain, thereby folding the E3 into an auto-inhib- Among the various HECT E3 ligases, a large variety of con- itory conformation . The NEDD4 subfamily can contain figurations are observed in the region located N-terminal to between two and four WW domains. The WW domains are the HECT domain. The human HECT E3 family consists of responsible for the recognition of substrates  and have 28 members of which 15 members can be categorized into also been found to form intramolecular interactions with the two subfamilies based on commonalities in the N-terminal HECT domain of the E3s . domains (Fig. 4). The human NEDD4 subfamily, charac- terized by the presence of WW and C2 domains, has nine HERC subfamily members and is the most prominent and well studied of the two families. The other family is the HERC E3 ligases The HERC subfamily is characterized by having one or more that consist of six members and have in common that they RCC-like domains (RLDs), an effector protein domain that contain one or more regulator of chromatin condensation was first identified in RCC1 . In humans, the HERC Fig. 3 Structural rearrange- ments in the catalytic HECT domain. Illustration of alternate HECT domain C-lobe positions as seen in the crystal struc- tures of UbcH7 (not shown HECT for clarity)-E6AP (green) (PDB ID: 1C4Z) and UbcH5b (salmon) ~ Ubiquitin (not shown HECT for clarity)-NEDD4.2 (cyan)(PBD ID: 3JW0). The flexible linker that allows for rearrangements of the N- and C-lobes with respect to each other is indicated. Catalytic cysteine residues are displayed as yellow balls. For details see text 1 3 Regulating the human HECT E3 ligases 3125 subfamily comprises six members, which can further be the ubiquitination activity and substrate specificity. HECT organized into two large and four small HERCs (Fig. 4). E3 adaptors associate with the E3 ligases by binding to HERC1 and HERC2 are large HERCs that contain two and domains in the N-terminal regions or to regions within the three RLDs, respectively. The small HERCs contain only HECT domain. one RLD. RLDs have dual functions as one side of the A classic example of a HECT adaptor is the viral protein domain acts as a guanine nucleotide exchange factor (GEF) E6, which was the protein that led to the discovery of the for the small GTPase Ran and the opposite side interacts HECT E3 ligases [6–8]. E6 is one of the two viral proteins with chromatin through histones H2A and H2B [43, 44]. expressed in HPV (human papilloma virus)-positive cervi- Through their interactions with histones, HERC E3 ligases cal carcinomas, the other being E7, which also utilizes the participate in various processes at the chromatin and in the cell proteasomal system to inactivate its targets . The nucleus (reviewed in ). two viral proteins each have an enormous impact on host gene expression patterns, and their oncogenic role is mainly “Other” HECT E3s attributed to their inactivation of tumor suppressors p53 and retinoblastoma (pRb) [7, 11, 51, 52]. The E7 oncoprotein is E6AP, the founding member of the family of HECT E3 thought to target the pRb tumor suppressor using the cullin 2 ligases, is profoundly impactful on the regulation of the ubiquitin ligase complex; however, this mechanism remains cell and has been extensively studied. Located at the N controversial [53, 54]. With regards to the E6 oncoprotein, it terminus (residues 24–87), E6AP harbors a zinc-binding is clear that it hijacks E6AP by binding to the LxxLL motif fold called the AZUL (amino-terminal Zn-finger of Ube3a located in the N-terminal domain of E6AP and utilizes the ligase) domain of which mutations have been associated activity of its HECT domain to ubiquitinate p53, thereby with Angelman syndrome . Another notable feature marking it for degradation by the 26S proteasome  of the E6AP protein is an LxxLL motif (where x denotes (Fig. 5a). Recent structural analysis has revealed that the any residue) located in the center of the protein (residues binding pocket for the LxxLL motif on E6 is formed by two 379–395), which is the binding site of E6  (for details, zinc domains and a linker helix . Other than redirecting see “adaptor proteins and co-activators”). Although not stud- E6AP substrate specificity to ubiquitinate p53, the E6/E6AP ied as thoroughly as E6AP, the members HUWE1, UBR5, complex also ubiquitinates other cellular proteins, the degra- TRIP12, and HACE1 have also been subject to a signifi- dation of which contributes to E6-induced cellular immor- cant amount of research (reviewed in ). HUWE1 is a talization or transformation . These include the TERT giant 4374 amino-acid residue protein that contains a WWE (telomerase reverse transcriptase) gene repressor NFX1-91, domain, a BH3 domain, an UBA domain, and Armadillo resulting in increased telomerase activity [57, 58], E6TP1 repeats (ARM). HUWE1 naturally targets the p53 tumor (E6 targeted protein 1) [59, 60], MCM7(mini chromosome suppressor for degradation, in contrast to E6AP which only maintenance protein 7) , and BAK (Bcl-2 homologous targets p53 once it is hijacked by the viral E6 protein . antagonist killer) . Consistent with this, knockout studies TRIP12 has also been implicated in the regulation of p53; of E6 and E6AP have shown that E6 relies virtually exclu- however, this regulation is indirect through the targeting of sively on E6AP to alter the cellular gene expression . p14ARF, a key regulator of p53 . Another commonality The alterations that E6 binding creates in the substrate between TRIP12 and HUWE1 is the presence of a WWE specificity of E6AP cannot always simply be explained by domain and Armadillo repeats. Other domains shared among the binding of E6 to substrates. Studies have shown that this family of HECT E3s are the UBA domain (UBR5 and p53 does not bind E6AP or the E6 adaptor in isolation from HUWE1), Ankyrin repeats (HACE1 and HECTD1), and IQ each other , whereas other studies showed only weak motifs (UBE3B and UBE3C). and insignificant E6–p53 interactions [65, 66]. Consistent with this, a recent analysis of the crystal structure of the E6/E6AP/p53 ternary complex showed that the p53 binding Substrate recruitment and catalytic activity cleft on E6 is formed upon binding of the LxxLL peptide regulation . Other substrates recruited to the E6/E6AP complex by E6 show that the viral protein does function in a manner Adaptor proteins and co‑activators expected from an adaptor protein. For example, the E6 pro- teins of ‘high-risk’ HPV have a C-terminal PDZ (named for A prominent mechanism that regulates the substrate-specific the proteins PSD95, DLG, and ZO1) domain-binding motif conjugation of ubiquitin is the recruitment of HECT E3s (PBM) through which they bind PDZ-domain-containing to their substrates by adaptor proteins. Assuming that the substrates irrespective of whether the E6 is in complex with HECT E3 is in an active state, adaptors proteins can recruit E6AP . Nineteen PDZ-domain-containing proteins have the E3 to its ubiquitination substrate, thereby contributing to been confirmed as binding partners of E6, of which several 1 3 3126 J. Sluimer, B. Distel 1 3 Regulating the human HECT E3 ligases 3127 ◂Fig. 4 Human HECT E3 protein domain architecture. Overview of that acts as an antagonist of the phosphatidylinositol-3-ki- the domain organization of human HECT E3 ligases. Protein domains nase (PI3K) signaling pathway by dephosphorylating the were predicted by the InterPro server . HECT E3 ligases are second messenger phosphatidylinositol 3,4,5-trisphosphate characterized by the presence of a conserved HECT (homologous (PIP ) to phosphatidylinositol 4,5-bisphosphate (PIP ) . to E6AP C-terminus) domain that is located at the C-terminus of 3 2 the proteins. The human HECT E3 ligase family is grouped into two Through its role in phosphatidylinositol homeostasis, PTEN subfamilies and 13 “other” HECT E3 ligases based on their domain is recognized as a pivotal tumor suppressor and regulator architecture N-terminal to the HECT domain. The NEDD4 subfamily of cellular processes including proliferation, survival, and comprises nine members each having an N-terminal C2 domain and migration . Several PY motif adaptors have been found between two and four WW domains. The HERC subfamily has six members that each contains between one and three RLDs (RCC-like to mediate PTEN ubiquitination including NDFIP1 (Nedd4 domains) while the two large family members, HERC1 and HERC2, family-interacting protein 1) and NDFIP2 and more recently additionally contain various other domains. The “other” HECT E3 NUMB. The NDFIP adaptors can recruit various NEDD4 ligases contain various domains as shown. Domain abbreviations 2+ E3s to target PTEN either for degradation or nuclear trans- used are as follows: C2 C2 domain (Ca -binding domain), WW WW domain, RLD RCC-like domain, SPRY B30.2/SPRY domain, WD40 location [84–86] (Fig. 5b). In response to ischemia, NDFIP1 W-D repeat domain, Cytb5 cytochrome b5-like heme/steroid-binding recruits NEDD4.1 or NEDD4.2 to mono-ubiquitinate PTEN, domain, DOC APC10/DOC domain, MIB MIB-HERC2 domain, which leads to translocation of PTEN to the nucleus , AZUL amino-terminal Zn-binding domain of UBE3A ligase, ARM where it controls processes not related to P IP hydrolysis Armadillo repeat-containing domain, UBA ubiquitin-associated 3 domain, WWE WWE domain, BH3 Bcl-2 homology 3 domain, ANK such as chromosome integrity and cell cycle progression Ankyrin repeat-containing domain, ZnF Zinc finger domain, PABC . By contrast, NDFIP-mediated recruitment of PTEN to polyadenylate-binding protein C-terminal domain, IQ IQ motif/ the HECT E3 WWP2 results in PTEN poly-ubiquitination EF-hand binding site, PHD PHD-type zinc finger, Filamin filamin/ and its subsequent degradation . These observations ABP280 repeat-like domain suggest that the HECT E3 that is recruited by the adaptor determines the outcome for the substrate. WWP2 likely are known as tumor suppressors [69, 70]. Experiments ubiquitinates PTEN with Lys48 and/or Lys11-linked chains with cell and mouse models show that the PBM is impor- that are commonly associated with proteasomal degradation, tant for carcinogenesis [71–74] and that this is reliant upon whereas NEDD4.1 has been shown to mono-ubiquitinate proteolytic targeting of PDZ-domain-containing substrates PTEN , a modification often associated with non-prote- by the E6/E6AP complex [75–77]. This is consistent with olytic functions. Intriguingly, recruitment of NEDD4.1 by the observation that all cancer-causing HPV types contain a different adaptor, NUMB, results in PTEN poly-ubiqui - a PBM, whereas low-risk HPV types often do not contain a tination and degradation , suggesting that the adaptor PBM (reviewed in ). may also influence the type of ubiquitin chain catalyzed The binding of E6 to E6AP does not only affect its by the HECT E3. Another NEDD4 member regulated by substrate recognition, but also the catalytic activity of its NDFIP1 is ITCH, an E3 ligase that targets the transcription HECT domain is increased. Consequently, its regular sub- factor JunB for degradation in a way reminiscent to that of strate proteins are also increasingly ubiquitinated by the E6/ the recruitment of WWP2 to the substrate PTEN. Indeed in E6AP complex . Notably, a similar allosteric interaction, the absence of NDFIP1, ITCH cannot bind JunB to ubiqui- which enhances E6AP activity, has been seen with HERC2. tinate it, resulting in the accumulation of JunB. Consistent HERC2, another HECT E3, binds to E6AP in a region on with this, the absence of NDFIP1 has been shown to cause the N terminus (residues 150–200) and thereby increases inflammation in mice as a result of JunB accumulation . the catalytic activity of E6AP . Together these studies Interactions of PY-containing adaptors with the WW show that E6 is a versatile adaptor that recruits substrates to domains of HECT E3s are also seen for viral proteins the E6AP complex and additionally increases the catalytic (Fig. 5c). The viral protein VP40, which is found in the filo - activity of the HECT domain through allosteric interactions. viruses Ebola (eVP40) and Marburg (mVP40), contains a It should not come as a surprise that viruses utilize adap- proline-rich PPxY motif that it utilizes to hijack NEDD4.1 tors to control HECT E3 ligases, as the cell also commonly through binding of its third WW domain [89, 90]. The uses them to regulate HECT E3s. Some of the most exten- release of viral particles is dependent on the binding of VP40 sively studied cellular adaptors are those that interact with to NEDD4.1, thereby hijacking its ubiquitination activity to the WW domains of the NEDD4 subfamily. Adaptors of mediate virus budding . A recent study also showed that NEDD4 typically contain PY motifs (PPxY or LPxY, x is the VP40 proteins hijack ITCH to regulate the budding pro- any residue) that interact specifically with WW domains [80 , cess . Given the HPV viral protein E6 hijacking E6AP 81]. One informative example of a target that is recruited to suppress p53 and VP40 hijacking NEDD4 family E3s to NEDD4 E3 ligases through interaction with PY motif- to mediate virus budding, it is ostensible that HECT E3s containing adaptors is PTEN (phosphatase and tensin are desirable targets which viruses can use to control their homolog). PTEN is a plasma membrane lipid phosphatase host cells. Hence, inhibiting these HECT E3–viral protein 1 3 3128 J. Sluimer, B. Distel Fig. 5 Modulation of HECT E3 ligase function by adaptor proteins E3 ligases of the NEDD4 subfamily to their substrate. In response and co-activators. a Binding of the HPV E6 protein to a conserved to ischemia, NDFIP1 recruits NEDD4 to mono-ubiquitinate PTEN, sequence (LxxLL motif, not shown) in the N-terminal domain of resulting in translocation of PTEN into the nucleus. Recruitment of E6AP allows the E6/E6AP complex to recruit the tumor suppressor WWP2 HECT E3 ligase by the NDFIP1 adaptor results in poly-ubiq- protein p53. E6AP-dependent ubiquitination of p53 targets the protein uitination of PTEN, thereby targeting PTEN for proteasomal degrada- for proteasomal degradation, thereby promoting HPV-induced cer- tion. c The viral VP40 protein hijacks NEDD4.1 thereby promoting vical carcinogenesis. Not shown are the other targets inactivated by ubiquitination of viral proteins and stimulating the viral budding pro- the E6/E6AP complex whose degradation may contribute to carcino- cess genesis. b NDFIP1, a PY motif-containing adaptor, recruits HECT interactions, for example, with small molecules can be of an adaptor for SMURF1 and SMURF2 by recruiting these therapeutic relevance. Indeed various compounds have HECT E3s to their substrate the TGF-β receptor . Simi- been found that inhibit the interaction between the Ebola lar to the mechanism of NDFIP1- and NDFIP2-mediated and Marburg virus VP40 proteins and NEDD4 HECT E3s recruitment described previously, SMAD7 has PY motifs . Although these compounds may inhibit interactions that interact with the WW domains of the SMURF E3s. In of NEDD4 with any PPxY motif-containing protein, they addition to its function as an adaptor, SMAD7 also has a are still attractive potential antiviral agents. Screens for function to activate the HECT domain. SMAD7 recruits small molecule inhibitors have also been successfully done UbcH7, the E2-conjugating enzyme of SMURF2, thereby to find compounds that inhibit the interaction between E6 enhancing the ubiquitin ligase activity of the E3 . To and E6AP . Several of these inhibitory compounds were facilitate E2–E3 interaction, SMAD7 binds the HECT shown to block p53 degradation in HPV-infected cells. Fur- domain of SMURF2 and binds UbcH7 with its N-terminal ther study of these compounds may lead to the development domain (Fig. 6a). Analysis of the E2-binding domain of of beneficial therapeutics. SMURF2 suggested that SMURF2 has an inherent low an ffi - ity for its E2-conjugating enzyme. This low E2–E3 binding Regulation by recruitment of E2‑conjugating affinity suggests that the E3 enzyme is dependent on other enzymes proteins for optimal interaction with its E2-conjugating enzyme and thus its ubiquitination activity. HECT ubiquitination activity is also regulated at the level In addition to the recruitment of E2s by adaptors, inhibi- of recruiting E2-conjugating proteins. SMAD7 functions as tors that prevent the HECT E3s from interacting with 1 3 Regulating the human HECT E3 ligases 3129 Fig. 6 Regulation of E3-E2 interactions. a Binding of SMAD7 to HECT and the E2, thereby enhancing E2 binding and ubiquitin ligase SMURF2, through interaction of its PY motifs with one of the WW activity. b Non-covalent binding of interferon-stimulated gene 15 domains in SMURF2, not only relieves the auto-inhibitory conforma- (ISG15) to NEDD4.1 blocks interaction of the HECT E3 with its tion of the HECT E3 (see Fig. 7b) but also stimulates E2 binding: the E2-conjugating enzyme, resulting in reduced E3 ligase activity amino-terminal domain (NTD) of SMAD7 contacts both the SMURF E2-conjugating enzymes can also regulate HECT activ- have a default state of auto-inhibition. Although the domain ity. The interaction of NEDD4.1 with its E2-conjugating architecture of the NEDD4 family is similar, the mecha- enzyme is negatively regulated by interactions with ISG15, nisms of auto-inhibition vary [39, 100, 101]. Two exem- an ubiquitin-like protein. The resulting decrease in NEDD4 plary mechanisms of auto-inhibition can be found in ITCH E3 ligase activity causes a reduced ubiquitination of the and SMURF2 (Fig. 7). The auto-inhibited state of ITCH is Ebola virus VP40 (discussed in the previous section), acquired through the binding of its WW domain region to its thereby blocking viral budding [97, 98] (Fig. 6b). ISG15 is HECT domain (Fig. 7c) . Recent structural analysis has an interferon-stimulated gene that can be present in the cell revealed further details of the auto-inhibitory conformation in free form or covalently bound to a substrate as a result of ITCH: the interface of the auto-inhibited ITCH involves of an enzymatic process called ISGylation . ISGyla- the second WW domain (WW2) and a linker region connect- tion of NEDD4.1 is not necessary to obtain the inhibitory ing WW2 and WW3 . The WW3 domain may still be effect on NEDD4.1; non-covalent interactions of ISG15 with relevant for auto-inhibition as previous research established NEDD4.1 are sufficient to prevent the E2–E3 interaction. that mutations in either WW2 or WW3 resulted in activation of ITCH . The importance of the WW2–WW3 linker Inter‑ and intramolecular interactions of HECT E3 region in regulating E3 ligase activity was recently con- ligases firmed for WWP2, a HECT closely related to ITCH . Indeed, Chen et al. establish that a linker region connecting HECT E3 ligases are often regulated by intramolecular (i.e., the WW2 and WW3 domains is necessary to lock the E3 in within the same molecule) or intermolecular (i.e., with other its auto-inhibitory conformation. The SMURF2 inhibitory identical proteins) interactions. Through these intra- and state is acquired similarly, but instead of the WW domain intermolecular mechanisms, the HECT E3s can control the region it is its C2 domain that binds to the HECT domain activity of their own catalytic domain. Often these mecha- (Fig. 7b) . Similar to SMURF2, interactions between the nisms determine the default state of the HECT protein and, C2 and HECT domains also inhibit SMURF1 (Fig. 7a). In thus, regulatory proteins that change these inter- and intra- contrast, SMURF1 inhibitory interactions are not intramo- molecular interactions can control the activity of the HECT lecular. SMURF1 is not able to fold into an intramolecular E3. auto-inhibitory state likely due to the shorter nature of the protein. Instead, the C2-HECT domain auto-inhibitory inter- Interactions of N‑terminal domains with the HECT domain actions of SMURF1 proteins are facilitated by the formation of homodimers . Some HECT E3s can arrange themselves into inactive con- The regulatory mechanisms that the cell uses to control formations by interactions of the N-terminal region with the HECT E3s often revolve around relieving previously dis- HECT domain. Several of the WW and C2 domains of the cussed auto-inhibitory interactions. Intuitively, a wide vari- NEDD4 E3s can interact with their corresponding HECT ety of mechanisms can be envisioned that relieve these auto- domains, thereby blocking access to the catalytic site. Due to inhibitory and homo-oligomeric interactions. The previously these conformations, it is common for NEDD4 E3 ligases to mentioned examples of ITCH, SMURF1, and SMURF2 1 3 3130 J. Sluimer, B. Distel Fig. 7 Modes of relief of auto-inhibitory interactions of the NEDD4 ing and ubiquitination of SMURF1 substrates. b Binding of SMAD7 subfamily. Various mechanisms are known to relieve auto-inhibitory to WW domains of SMURF2 relieves auto-inhibition and enhances interactions between HECT and C2/WW domains. SMURF1 forms E2 binding, thus stimulating E3 ligase activity (see also Fig. 6a). an auto-inhibited homodimer through intermolecular interaction of Serine/threonine phosphorylation of ITCH in a proline-rich region the C2 domain of one monomer with the HECT domain of the other between the C2 and WW domains (c) or tyrosine phosphorylation of (a), while in all other known NEDD4 subfamily members the auto- the NEDD4.1 C2 domain (d) relieves auto-inhibition. e The increase 2+ inhibitory conformation is mediated by intramolecular interactions in Ca concentration in the cell disfavors the NEDD4.2 C2-HECT involving C2 or WW domains and the HECT domain (b–e). a Allos- domain contacts, thereby relieving the auto-inhibitory conformation. 2+ teric interactions of various adaptor proteins with SMURF1 disrupt In the presence of Ca , the C2 domain can bind PIP (phosphati- the SMURF1 auto-inhibited homodimer and promote substrate bind- dylinositol 4,5-bisphosphate) head groups in the membrane or IP ing. CDH1 (Cadherin 1) and CKIP1 (Casein kinase-2 interacting (inositol 1,4,4-triphosphate) molecules in the cytoplasm, which may protein 1) may work sequentially: interaction of CDH1 with the C2 in turn determine the intracellular localization of the HECT E3 ligase. domain disrupts the SMURF1 homodimer, subsequent binding of L linker region connecting the WW2 and WW3 domains. For details, CKIP1 to the linker region between the WW domains promotes bind- see text inhibitions also provide good examples of the various is required for disruption of the auto-inhibited SMURF1 mechanisms that relieve inhibition (Fig. 7). SMURF2 auto- homodimer, CKIP1 promotes binding and ubiquitination inhibition is relieved by the binding of its substrate SMAD7 of SMURF1 substrates. The auto-inhibitory conformation to the WW domain adjacent to the HECT domain (Fig. 7b) of ITCH is relieved by phosphorylation (Fig. 7c). In this . Disruption of the SMURF1 homodimer is achieved instance, ITCH is phosphorylated at residues S199, S232, by allosteric interactions of several adaptor proteins includ- and T222, all of which are located in a proline-rich region ing CKIP (casein kinase-interacting protein), which binds (PRR) between the C2 and WW domains . PY motif- to the WW domain ; CDH1 (Cadherin 1), which binds containing interactors such as NDFIP1 and NDFIP2 can to the C2 domain ; or CCM2 (cerebral cavernous mal- also release auto-inhibition through their interactions with formations 2), which binds to the HECT domain (Fig. 7a) WW domains . To release auto-inhibitory conforma- . Recent evidence suggests that CKIP and CDH1 have tions involving the WW-linker-binding interface such as in different roles in SMURF1 activation . While CDH1 ITCH, the PY motif-containing co-activators are likely to 1 3 Regulating the human HECT E3 ligases 3131 rely on multiple PY–WW interactions able to disrupt the 111]. However, the inhibitory interactions of Rsp5 are sub- auto-inhibitory WW-linker to HECT binding. This linker stantially different from the previously discussed inhibitory region may also be directly targeted to relieve auto-inhibi- interactions of NEDD4 ligases between the C2 and HECT tion. The WW2–WW3 linker contains tyrosine phosphoryla- domains. Rsp5 is proposed to utilize an oligomerization tion sites, and experiments mimicking phosphorylation of interface in its HECT domain to form inhibitory trimers. these sites in WWP2 showed increased activity of the HECT Interestingly, the experiments of Attali et al. suggest that . The linker regions connecting the various N-terminal the accessibility of this trimerization interface is con- domains of HECT E3 ligases may have a more impactful trolled by the ubiquitination of specific lysine residues in an role on HECT regulation than previously anticipated. Chen α-helical segment N-terminal to the HECT domain, called et al.  discuss a linker region C-terminal of the WW1 the α1-helix . The HECT ubiquitin-binding domain domain in NEDD4.1 that has an α-helical structure simi- (HECT-UBD), also known as the ‘exosite’ [112, 113], has an lar to the WW2–WW3 linker in WWP2. Indeed deletion of important role in this regulatory mechanism as its pull on the this linker region in NEDD4.1 resulted in increased auto- ubiquitin conjugated α1-helix is suggested to lead to a con- ubiquitination activity. In addition, tyrosine phosphorylation formational change that exposes the trimerization interface has been shown to relieve the auto-inhibitory interactions of . The resulting trimerization shuts down the catalytic NEDD4.1 (Fig. 7d). NEDD4.1 is phosphorylated at a tyros- activity of the HECT domain. Conservation analysis of the ine residue in the C2 and in the HECT domain by c-Src α1-helix domain suggested that this oligomerization mecha- kinase . nism is also prevalent among human NEDD4 ligases. Exper- NEDD4.2 is regulated by its C2 domain through another iments using NEDD4.1 with a ubiquitin-fused α1-helix and K523,525R distinct mechanism (Fig. 7e). The auto-inhibited form of a NEDD4.1 mutant that cannot be ubiquitinated at NEDD4.2 is stabilized by the interaction of its C2 domain its α1-helix are consistent with this . Ubiquitin-fused 2+ with its HECT domain. Increased Ca concentration in the NEDD4.1 results in its oligomerization and diminished K523,525R cell leads to the activation of NEDD4.2 by disrupting this ubiquitination activity, whereas NEDD4.1 did not auto-inhibitory conformation . The HECT-binding form oligomers and exhibited increased activity. Thus, ubiq- 2+ site on the C2 domain is shared with the Ca binding site, uitination of the α1-helix may be an important regulatory 2+ suggesting that the Ca interactions cause repulsion of the mechanism to control the activity of various NEDD4 ligases. HECT domain. In addition to the activation of NEDD4.2, As NEDD4.1 auto-ubiquitinates the α1-helix, a deubiquit- these interactions may also be involved in the localization inating enzyme may control the activity of the E3. It may of the HECT E3 ligase. NEDD4.2 can be relocated through be that the α1-helix ubiquitin-mediated oligomerization interactions of its C2 domain with the head group of phos- is as common as the C2-HECT domain oligomerization, phatidylinositol 4,5-bisphosphate (PIP ) and its second where the former is controlled by the ubiquitin status of the messenger inositol 1,4,5-trisphosphate (IP ), which is the α1-helix and the latter by phosphorylation or Ca binding. soluble version of the PIP head group. The interactions of 2+ PIP and IP are facilitated by Ca ions that form a bridge Activating E6AP–E6AP interactions 2 3 to the C2 domain. PIP prefers localization at the cell mem- brane, whereas IP prefers to diffuse throughout the cyto- Oligomerization may also play a role in the activa- plasm. The PIP /IP ratio can determine the distribution of tion of HECT E3s rather than their inhibition. E6AP 2 3 NEDD4.2 as localization of the E3 depends on which of the trimerization has been reported to promote its ubiqui- two molecules it associates with . These results may tin ligase activity . Early evidence for coopera- suggest phospholipase C (PLC) as an important upstream tive E6AP–E6AP interactions were already suggested, 2+ regulator of NEDD4.2 as PLC triggers the increase of Ca because E6AP was only found to efficiently auto-ubiqui- in the cytoplasm and controls the ratio of PIP /IP through tinate in the presence of other E6AP enzymes, suggesting 2 3 the hydrolysis of PIP . Since all NEDD4 ligases contain a intermolecular transfer of ubiquitin . In support of 2+ C2 domain, the discussed regulation through Ca may be an oligomeric structure of E6AP, crystallographic analy- more common among this HECT E3 family. sis of its HECT domain revealed a trimeric arrangement that is stabilized through N-lobe/N-lobe interactions Ubiquitin‑binding domain controlled oligomerization . However, the trimeric form of E6AP was initially dismissed as the HECT domain construct is monomeric Formation of intermolecular interactions between identi- in solution and a mutation in the trimerization interface cal HECT E3 ligases has also been studied in the NEDD4 (F727A) did not affect the ability of the enzyme to trans- yeast ortholog Rsp5. Similar to SMURF1, Rsp5 does not fer ubiquitin from E2 to its active site . Rather than appear to be inhibited by intramolecular interactions , forming oligomeric complexes, the interactions between but rather through the formation of Rsp5 oligomers [110, E6AP were proposed to be transient and the trimeric form 1 3 3132 J. Sluimer, B. Distel of the E6AP HECT domain in the crystal was attributed domain as well as if/how intra- and intermolecular interac- to a crystallization artifact. However, recent biochemical tions impact E3 ligase function in the proposed trimers. experiments showed that GST (glutathione S-transferase)- fused E6AP has an increased poly-ubiquitin chain for- HUWE1 oligomerization and auto‑inhibition mation in vitro compared to the un-fused E6AP. These observations suggested that GST–GST interactions  HUWE1 is a HECT E3 that can be down-regulated through promote E6AP oligomerization resulting in increased a combination of oligomerization and auto-inhibitory inter- activity, which prompted further research into the role actions . HUWE1 forms an auto-inhibitory homodi- of E6AP oligomerization . Kinetic and biophysical mer where both inter- and intramolecular interactions are experiments with the un-fused, full-length, E6AP pro- involved in the inhibition. In this case, a crystal structure vided stronger evidence for an activating role of E6AP of the C-terminal domain of HUWE1 revealed an asym- oligomerization, as E6AP trimers were found to be more metric dimer that is stabilized through hydrophobic interac- active than their monomeric counterparts. In support of tions mediated by a region adjacent to the HECT domain. this, mutation of a key residue in the trimerization inter- HUWE1 counteracts its own inhibition through an intra- face of E6AP, F727, which binds to a hydrophobic pocket molecular interaction with a segment located 50 residues of the adjacent subunit, substantially decreased the cata- upstream of the dimer-binding region. The HUWE1 inhibitor lytic activity of E6AP using polyubiquitin chain forma- and tumor suppressor p14ARF have been shown to bind and tion as a functional readout. The E6AP trimer harbors a disable this ‘activation segment’, thereby promoting inhibi- second subunit interface important for the stabilization of tory dimerization. The involvement of the p14ARF inhibitor the trimer [31, 114, 117]. The second interface is located suggests that, in contrast to the previously discussed oli- further from the catalytic site. Interactions at this second gomerization mechanisms, the inhibitory conformation of interface were shown to be regulated by tyrosine phos- HUWE1 is not its default state. Structural analysis of the phorylation , which is discussed more elaborately asymmetry of the HUWE1 dimer is intriguing, as it appears in the chapter of post-translational modifications. Muta- to affect the activity status of the two HECT domains. The tion of key residues at this second subunit interface also HECT activity of one subunit in the dimer is impaired by reduce the catalytic activity of E6AP , further sup- a conformational lock of the C-lobe and blocked access to porting the notion that the fully active form of E6AP is the catalytic site through interactions with the other subu- a trimer. nit. This latter subunit may be inhibited through allosteric Remarkably, none of the other crystallized HECT interactions in the dimer; however, there is no indication domains forms a crystallographic trimer (reviewed in [16, that the flexibility of the C-lobe of this subunit is restricted, 118]), suggesting that trimerization is a unique feature of so its HECT domain may still be catalytically relevant (for the E6AP HECT domain. However, the crystallized E6AP details see [16, 119]). It appears that HUWE1 has a unique HECT construct lacks an α-helix segment N-terminal of oligomerization mechanism in contrast to the previously dis- the HECT domain (α-1 helix; previously discussed to cussed more common oligomerization mechanisms. regulate Rsp5 oligomerization) that is present in all other crystallized HECT domain constructs. In these structures Post‑translational modifications of HECT E3 ligases the α-1 helix shields the hydrophobic pocket otherwise occupied by F727, suggesting that the presence of the α-1 Various post-translational modifications of HECT E3 ligases helix in these HECT domain constructs may prevent trim- are known to influence catalytic activity. Phosphorylation erization. In support of a role of the α-1 helix in regulating and ubiquitin-like modifications are types of post-transla- E6AP oligomerization, Ronchi et al.  showed that tional modifications that commonly regulate various HECT the addition of a peptide corresponding to the α-1 helix E3s. Post-translational modifications can influence HECT promoted the dissociation of the E6AP trimer and strongly activity by causing conformational changes in the E3 ligases reduced its E3 ligase activity in vitro. It is remarkable that or by influencing interactions of HECT E3s with adaptors or trimerization of E6AP and Rsp5 seems to have opposing other regulatory proteins. outcomes, namely activation of the former and inactivation of the latter. Although modeling has suggested that Rsp5 Phosphorylation (and possibly other NEDD4 family members) uses similar interfaces as E6AP to form trimers , further structural As previously discussed, phosphorylation of a PRR of ITCH analysis is required to confirm this hypothesis. In addition, can activate the HECT domain by relieving ITCH from its there is currently no structural information available on the auto-inhibitory fold (see Fig. 7c). More specifically, ITCH full-length proteins. Therefore, it remains unclear what is phosphorylated on three residues (S199, T222, S232) of the contribution is of the regions N-terminal to the HECT a PPR residing in between the C2 and the first WW domain 1 3 Regulating the human HECT E3 ligases 3133 by the JNK1 serine/threonine kinase [101, 120]. Recruit- promote its E3 ligase activity, it is conceivable that Y636 ment of JNK1 to ITCH is facilitated by an interaction of phosphorylation regulates E6AP by deterring its ability to a D domain-like sequence within the N-lobe of the HECT oligomerize. However, direct evidence for this is lacking. domain. D domains consist of a group of basic residues fol- Mutants of E6AP that influence its phosphorylation are lowed by a cluster of hydrophobic residues and have been not exclusive to those introduced with gene editing. A muta- shown to recruit MAP kinases . Phosphorylation of tion of threonine 485 to alanine in E6AP was identified in a the PRR region weakens the interaction between the WW whole-exome sequencing study aimed at identifying de novo domain region and the HECT domain, thereby disrupting mutations linked to autism . Detailed analysis of the the auto-inhibitory conformation of ITCH . The PRR T485A mutation led to the discovery that T485 is phospho- of ITCH is the only known phosphorylation site of JNK1 rylated by PKA (protein kinase A) as a mechanism to regu- in HECT E3s so this mechanism may be unique to ITCH. late E6AP E3 ligase activity . The substitution of threo- NEDD4.1 is another example where the auto-inhibition nine for an alanine makes it impossible to phosphorylate of a HECT E3 is relieved by phosphorylation (Fig. 7d). The the residue and thereby disables phosphorylation control of T485A mechanism varies from the previously discussed type of E6AP. In HEK293T cells, it was shown that the E6AP NEDD4 family phosphorylation in that NEDD4.1 is phos- mutant actively auto-ubiquitinates, whereas a T485E mutant phorylated at tyrosine residues in the C2 (Y43) and HECT that mimics phosphorylated E6AP was inhibited for auto- domain (Y585), and this phosphorylation is catalyzed by ubiquitination. In addition to reducing auto-ubiquitination, the c-Src kinase . The phosphorylated C2 and HECT phosphorylated E6AP was also shown to have repressed E3 domains of NEDD4.1 do not form the auto-inhibitory con- ligase activity towards substrates such as hHR23A using formation most probably due to electrostatic repulsions in vitro analysis with the same mutants. Interestingly, the caused by the phosphate groups. SYK-mediated tyrosine phosphorylation at T485 not only affects E3 ligase activity phosphorylation of HUWE1 is another example of how but also the ability of E6AP to self-associate. While wild- phosphorylation can disrupt auto-inhibition . Unlike type E6AP and the phospho-mimetic T485E mutant showed the previously discussed mechanism where addition of a no self-association, the T485A mutant was able to interact phosphate moiety disrupts auto-inhibition, phosphorylation with itself . Whether the observed self-association of of HUWE1 causes the disassociation of HUWE1 from its the T485A mutant is related to the crystallographic E6AP inhibitor p14ARF. As discussed previously, the release of trimer remains to be elucidated. A recent follow-up study on T485A p14ARF enables HUWE1 to form an intramolecular con-the E6AP mutant revealed that its expression results in formation that counteracts the inhibitory dimer. increased Wnt (wingless-type MMTV integration site fam- One of the earliest cases reported of a HECT E3 regulated ily member) signaling through the ubiquitination of several by phosphorylation is that of NEDD4.2 . NEDD4.2 is proteasome subunits that are part of a distinct proteasome phosphorylated by the kinase SGK1 (serum/glucocorticoid- subdomain . The ubiquitination of proteasome subu- regulated kinase 1) which utilizes a PY motif that binds to nits by E6AP reduces proteasome subunit abundance and the E3s WW domains . Serine phosphorylation (S468) activity [128, 129], resulting in the accumulation of stable of NEDD4.2 promotes recruitment of the adaptor protein β-catenin in the nucleus and subsequent activation of the 14-3-3 to the phosphorylated NEDD4.2, thereby reducing Wnt signaling pathway . Together, the studies on the T485A the interaction of NEDD4.2 with its natural substrate ENaC phospho-resistant E6AP show how the phosphorylation (Epithelial Na Channel), leading to increased ENaC surface of one residue in a HECT E3 can control crucial cellular expression. processes. Phosphorylation has also been shown to negatively regu- late the E3 ligase activity of E6AP . E6AP is tyrosine Ubiquitination/deubiquitination phosphorylated by c-Abl at residue 636 within its HECT domain. Mutation analysis of Y636 suggests that this residue Ubiquitination is another PTM that can regulate HECT E3 controls the E3 ligase activity in a substrate-specific manner. ligases. HECT E3 ligases may be ubiquitinated by other E3 This substrate specificity was shown for the E6AP phos- ligases or ubiquitinate themselves in a process called auto- phorylation resistant mutant Y636F, which has impaired ubiquitination. Often the ubiquitination of HECT E3 ligases E3 ligase activity towards the substrate hHR23A (human functions as a mechanism of down-regulation by marking homolog of Rad23), although the ubiquitination of p53 in them for proteasomal degradation . The regulatory the presence of HPV E6 protein remained similar to that effects of ubiquitin on HECT E3s are often controlled at the of wild-type E6AP. Analysis of the E6AP crystallographic level of deubiquitination. trimer suggested that the Y636 is present in the second subu- One HECT E3 that is negatively regulated by ubiquitina- nit interface important for stabilization of E6AP oligom- tion is TRIP12. TRIP12 is ubiquitinated and thereby targeted ers. Since the oligomerization of E6AP has been shown to for proteasomal degradation. The control of this regulatory 1 3 3134 J. Sluimer, B. Distel degradation has been attributed to the DUB (deubiquitinat- the TGF-β pathway. SUMOylation of SMURF2 is facili- ing enzyme) USP7 . USP7 associates with TRIP12 tated by the SUMO-E2-conjugating enzyme UBC9 and and stabilizes it by deubiquitinating the HECT E3. This the SUMO E3 ligase PIAS3, which target distinct lysine USP7–TRIP12 interaction has been shown to lead to the residues of SMURF2. In a follow-up study, it was shown ARF ubiquitination of the tumor suppressor p14 . Unsurpris- that SMURF2 SUMOylation suppresses the invasiveness ingly, overexpression of USP7 and TRIP12 has been found of breast cancer organoids . The mechanism through to be associated with poor prognosis in cancers with aber- which SUMOylation regulates SMURF2 activity has not yet ARF rant expression of p14 , such as hepatocellular carcinoma been determined. However, the location of the lysine resi- (HCC) . Interestingly, USP7 itself is also targeted by dues SUMOylated by PIAS3, in the C2 domain (Lys26) and TRIP12 for proteasomal degradation, indicating that they next to the C-terminal WW domain (Lys369), may suggest are mutual substrates for each other . Thus, USP7 which regulatory mechanisms of the HECT E3 are altered. increases the stability of TRIP12, whereas the TRIP12 does Proximity of the Lys369 SUMOylation site to the WW the opposite for USP7. Their effectiveness in targeting their domains may affect the binding of these domains to the PY cognate substrates relies on the homeostatic balance of the motifs of adaptor proteins, while SUMOylation of Lys26 two proteins. may be involved in the formation or disruption of the auto- E6AP is another example of a HECT E3 ligase that is inhibitory conformations mediated by interactions between regulated by ubiquitination, serving as a substrate for UBR5, the C2 and HECT domain. another HECT E3 ligase . UBR5 negatively regulates E6AP abundance, and as a result the loss of UBR5 leads NEDD8 modification to higher levels and a longer half-life of E6AP. While this regulatory effect does occur in uninfected cells, most of the Neddylation, the covalent binding of NEDD8 onto a target regulatory effects of UBR5 on E6AP are associated with protein, is another ubiquitin-like modification that has been HPV-infected cells. UBR5 interacts strongly with the HPV shown to regulate HECT E3 ligases. NEDD8 is 58% iden- protein E6 and UBR5 impairs the ability of the E6/E6AP tical to ubiquitin and is covalently bound to its substrates complex to target its substrates for degradation. in a process analogous to ubiquitination, using dedicated Ubiquitination by the yeast HECT E3 ligase, Rsp5 is E1 (NAE) and E2s (Ube2M and Ube2F) for activation and negatively regulated through an interaction with the Ubp2 transfer [136, 137]. Neddylation is a post-translational modi- deubiquitinating enzyme . When in complex with the fication primarily known for regulating cullin-RING ligases HECT E3 ligase, Ubp2 antagonizes the ubiquitination by (CRLs), a superfamily of RING E3 ligases . The cullin Rsp5 by catalyzing the opposing reaction. In this case, the subunit of the CRL complex needs to be neddylated on a ubiquitin-associated (UBA) domain-containing protein Rup1 single conserved lysine residue close to its RING-binding mediates the coupling of Ubp2 to Rsp5. The UBA domain site to allow recruitment of the E2 critical for ubiquitin liga- of Rup1 is not necessary for the interaction between Rsp5 tion activity . Various non-CRL neddylation targets and Ubp2; however, the UBA domain does stimulate Ubp2 have been proposed and are being investigated including deubiquitinating activity. The coupling of Rsp5 to the DUB the HECT E3 SMURF1 [14, 140]. SMURF1 catalyzes its is not necessarily a general negative regulator of Rsp5 ubiq- own covalent attachment of NEDD8 to activate its ubiquitin uitination as it may have specificity towards the substrates it ligase activity . In contrast to the specific neddylation of deubiquitinates. This substrate specificity would allow Ubp2 a single lysine residue seen in CRL neddylation, SMURF1 to determine which Rsp5 substrates are ultimately ubiqui- neddylates multiple lysine residues in various domains. tinated. In addition, Ubp2 may play an important role in Whether neddylation activates SMURF1 through promot- determining the topology of ubiquitin chains catalyzed by ing E2–E3 interactions similar to CRL activation or through Rsp5. Deubiquitination can limit the extension of ubiquitin other mechanisms remains to be discovered. In addition to chains, thereby favoring mono-ubiquitination over poly- SMURF1, SMURF2 has also been shown to be neddylated ubiquitination of certain substrates. and this neddylation leads to SMURF2 ubiquitination and its sequential degradation . Non-covalent binding of SUMOylation NEDD8 has also been shown to promote ubiquitin ligase activity of SMURF1 and SMURF2 . The Smurf pro- A recent study showed that the conjugation of the small teins contain a conserved domain that binds NEDD8, and ubiquitin-like modifier (SUMO) to SMURF2 regulates the disruption of this binding inhibits ubiquitin ligase activity activity of the SMURF2 HECT domain . The SUMOyla- of the Smurf HECT E3s . A study of the other mem- tion of SMURF2 enhances its ubiquitination activity towards bers of the NEDD4 subfamily found that ITCH, NEDL1, its target TβR1 (transforming growth factor B receptor 1), and NEDL2 can also catalyze auto-neddylation in addition resulting in degradation and concomitant suppression of to the Smurf E3s . Exceptionally, ITCH is the only 1 3 Regulating the human HECT E3 ligases 3135 HECT that is neddylated on a single lysine residue, similar polyubiquitin chains, some HECT E3s have a processivity to the CRL E3s. ITCH also functions as a neddylation E3 site (also called the “exosite”) in the N-lobe that binds the for JunB, targeting it for sequential ubiquitination and deg- last ubiquitin of the growing chain to keep it in position radation . for catalysis of the next ubiquitin. The processivity site interacts non-covalently with ubiquitin utilizing a series of ISG15 predominantly hydrophobic residues to contact an interac- tion surface on ubiquitin that involves the canonical Ile44 The interferon inducible ISG15 is another ubiquitin-like hydrophobic patch [112, 148, 149]. Experiments using small protein that has been shown to regulate the activity of molecule inhibitors have demonstrated that the processiv- NEDD4.1. As discussed above, binding of free ISG15 ity of NEDD4.1 can be regulated by targeting the exosite to NEDD4.1 blocks the interaction of the E3 with its E2 . These small molecule inhibitors specifically bind to thereby inhibiting the ubiquitin ligase (Fig. 6b) . Nota- the processivity site, thereby disrupting the formation of bly, ISG15 expression is induced in response to viral infec- polyubiquitin chains. Consequentially, NEDD4.1 changes to tions and its inhibiting effect on NEDD4.1 is an effective a distributive E3 ligase resulting in an increased amount of counter mechanism to the virus. Similar to the NEDD8 substrates that are mono-ubiquitinated. Whether the modula- interaction with SMURF1 and SMURF2, ISG15 binds tion of NEDD4.1 by targeting its site of processivity is also NEDD4.1 through non-covalent interactions. It is possible utilized as a regulatory mechanism in the cell remains to be that NEDD4.1 is covalently modified by ISG15 (ISGyla- determined. Interestingly, however, a recent screen for ubiq- tion); however, the interaction with free ISG15 seems to uitin variants (UbVs) with increased affinity for the exosite be sufficient to inhibit the E3s ubiquitination activity . has provided useful insights as to how HECT domain activ- Whether post-translational modifications with ISG15 also ity can be regulated and modulated . Detailed biochemi- regulate HECT E3 ligases remains to be determined. ISGyla- cal analysis and HECT-UbV co-crystallization experiments tion has been shown to regulate non-HECT E3 ligases such revealed that UbV binding at the N-lobe exosite could modu- as Parkin . The ISGylation of Parkin is catalyzed by late E3 ligase activity through a variety of mechanisms. For the HECT HERC6, which ISGylates two lysine residues example, binding of the UbV to the NEDD4.2 and Rsp5 of Parkin resulting in the disruption of its auto-inhibitory exosite promoted the transfer of ubiquitin from E2 to E3, conformation. whereas other UbV interactions promoted ubiquitin transfer from E3 to the substrate. Another two UbVs were shown to modulate NEDD4.2 activity by decreasing processivity and Modulating HECT E3 activity increasing distributive multi-mono-ubiquitination. As the engineered UbVs are highly specific for one HECT E3  In addition to the variety of substrates HECT E3s can target by way of their design and selection process, their specificity specifically, there is also variety in the type of ubiquitin and may be useful to target and modulate a specific HECT E3 ubiquitin-like moieties conjugated. HECT E3substrates may ligase without affecting other HECT E3s in the cell. be mono-ubiquitinated or poly-ubiquitinated with additional To create certain ubiquitin polymers, HECT E3 ligases variation in the lysine linkages in the poly-ubiquitin chains. may also cooperate with other E3 ligases. For example, it HECT E3s have been shown to catalyze various poly-ubiqui- was shown that a yeast HECT E3 Ufd4 can team up with tin chains such as Lys48-linked Ub chains, Lys63-linked Ub the RING domain E3 ligase Ubr1 to poly-ubiquitinate its chains, or other linkage types [21, 22, 145, 146]. The result- substrates . The addition of the Ubr1 E3 to the UFD ing poly-ubiquitin chains can have different configurations complex resulted in increased processivity towards the Ufd4 ranging from linear to branched chains that each can consist substrates. Even though the preference to catalyze differ - of homotypic or mixed linkages. In addition to ubiquitin, ent linkage polyubiquitin chains has not been observed in ubiquitin-like modifiers such as NEDD8 and ISG15 can also this Ubr1–Ufd4 interaction the cooperation of different be catalyzed by HECT E3 ligases. UBL modifiers can even E3 ligases may be a good explanation of such ubiquitin be part of ubiquitin polymers , further emphasizing the polymers. wide variety of moieties that can possibly be conjugated by Phosphorylated ubiquitin has recently been shown to HECT E3s. Depending on the type of moiety, the substrate modulate HECT E3 ligase to alter the specific lysine link - can be committed to divergent fates or display altered func- age of the polyubiquitin chains it catalyzes. The HECT E3 tions. Importantly, various mechanisms exist that can regu- UBE3C normally forms ubiquitin chains with a mixture late HECT E3 ligases to determine what type of ubiquitin of both Lys29 and Lys48 linkages ; however, when it and UBL mono- or polymers they catalyze. uses ubiquitin phosphorylated at serine 20 it preferably An important determinant in the ubiquitin chain forma- poly-ubiquitinates its substrates with Lys48-linked chains tion is the processivity of the E3 ligase. To efficiently form . Phosphorylation of ubiquitin on S20 is found, among 1 3 3136 J. Sluimer, B. Distel other PTMs, in mammalian cells ; however, the physi- E6AP were shown to have opposing regulatory outcomes. ological significance of S20 phospho-ubiquitin is currently Oligomerization using a similar trimerization interface unknown. The physiological importance of other phospho- appears to lead to inhibition of Rsp5 and activation of ubiquitins such as S65-phosphorylated ubiquitin has been E6AP. To understand how HECT domain-mediated trim- demonstrated. Specifically, the generation of S65 phospho- erization can lead to these opposing outcomes in activity, ubiquitins by PINK1 (PTEN-induced putative kinase 1) it will be key to determine the structural and functional was shown to activate the RING-E3 parkin . Thus, it is contributions of the region N-terminal to HECT domain. conceivable that S20-phospho-ubiquitins are also utilized in HECT E3s are capable of catalyzing a variety of ubiq- the cell as a mechanism to regulate E3s and perhaps PTMs uitin polymers and can additionally catalyze the attach- of ubiquitin are more commonly involved in the regulation ment of the ubiquitin-like modifiers such as ISG15 and of HECT E3 ligases. NEDD8. The processivity of HECT E3 ligases may also be an important factor in determining the outcome for its substrates. Various studies discussed in this review pre- Summary and significance sented regulatory mechanisms that influenced HECT E3 ligase processivity. The lysine linkage of ubiquitin chains A wide variety of mechanisms regulates the substrate speci- is often determined primarily by the HECT E3; however, ficity and control catalytic activity of the HECT E3 ligases. regulators can modulate HECT E3s to alter the lysine link- The domains N-terminal to the HECT domain play a pivotal age they catalyze. Thus, substrates of HECT E3s can be role in determining substrate specificity, as they are impor - post-translationally modified with a wide variety of ubiq- tant interfaces that bind to adaptor proteins. Competitive uitin polymers that may have various functional outcomes. inhibitors or post-translational modifications often control For a full understanding of the functional consequences the interactions between adaptors and N-terminal domains, of HECT E3substrate ubiquitination, we need to not only thereby modulating HECT E3 ligase activity. identify these substrates, but also characterize the inter- The intermediate step of transferring ubiquitin from ubiquitin connectivity of their ubiquitin polymers. While the E2 to the active site of the HECT increases the control challenging, such an approach has become feasible due exerted by the E3 ligase regarding subsequent catalysis. This to the recent advances in targeted proteomics techniques contrasts to ubiquitination by RING E3s, where due to the . direct transfer of ubiquitin from E2 to the substrate control Regulation of HECT E3 ligases plays an important part of the reaction is largely determined by the E2-conjugat- in the determination of cell fates and as such disruption of ing enzyme, especially with regards to the linkage type of HECT E3 function is often implicated in disease. Under- ubiquitin chain formation. Consequently, mechanisms that standing the mechanisms that regulate HECT E3s will be control specificity and activity of HECT E3 ligases are of critical in the search for treatments that intervene with or increased importance in determining the fate of HECT E3 enhance HECT E3 functions. Various diseases have been substrates. E2-conjugating enzymes remain important reg- associated with HECT E3 function. For example, increased ulators of the HECT E3 ligases they cooperate with. The E6AP activity has been associated with autism and most example of the SMURF2–UbcH7 interaction illustrated cervical cancers [56, 126], whereas reduced activity of that the binding affinity of E2 to HECT E3 is an important E6AP has been implicated in Angelman syndrome [9, 10]. factor in determining the catalytic activity and processivity Regulating HECT E3s by targeting their upstream regulators of HECT E3s. Blocking of the E2–E3 interaction has also may be a promising strategy. For example, a study showed been shown to regulate HECT E3s as seen in the example of that stimulating PKA by using pharmacological agents was ISG15 binding to the E2-binding site of NEDD4.1. an effective method to turn down E6AP activity in neurons A recurring type of mechanism that controls HECT . While most strategies using pharmacological agents E3 ligases, especially that of the NEDD4 subfamily, is may focus on inhibition, in some cases activation or modula- the self-control of inter- and intramolecular interactions. tion of the HECT E3 might be a more appropriate strategy. Through interactions of the HECT domain with N-ter- The screen using ubiquitin variants (UbV) found various minal C2 or WW domains, NEDD4 family members can sites on the HECT domain that upon interaction with an take up auto-inhibitory folds or form homodimers that UbV resulted in activation or modulation of the HECT E3. block catalytic activity until an external regulator unlocks Perhaps pharmacological agents can be developed that tar- the HECT E3. Mechanisms that can relieve these auto- get the same activation or modulation sites as a therapeutic inhibitory and oligomeric forms such as post-translational strategy. The wide variety of regulatory mechanisms that modifications, allosteric protein–protein interactions, and control HECT E3s in the cell should serve as an indicator 2+ Ca interactions have been highlighted for the NEDD4 of the many possible avenues that can be taken in order to family in this review. The trimers formed by Rsp5 and manipulate the activity of HECT E3 ligases. 1 3 Regulating the human HECT E3 ligases 3137 Acknowledgements We thank Stijn Bossuyt for his comments and epithelial–mesenchymal transition in a sumoylation-regulated suggestions. This work was supported by Grants from The Nether- manner. Cell Death Differ 23:876–888. https://doi.or g/10.1038/ lands Organization of Scientific Research (NWO-Zon-Mw; 91216045), cdd.2015.152 the Angelman Syndrome Foundation (ASF), and the Jérôme Lejeune 16. Lorenz S (2018) Structural mechanisms of HECT-type ubiqui- Foundation to BD. tin ligases. Biol Chem 399:127–145. https ://doi.org/10.1515/ hsz-2017-0184 17. 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