cancers Review The HECT E3 Ligase E6AP/UBE3A as a Therapeutic Target in Cancer and Neurological Disorders Asia Owais 1, Rama K. Mishra 2,3 and Hiroaki Kiyokawa 1,* 1 Department of Pharmacology, Northwestern University, Chicago, IL 60611, USA; [email protected] 2 Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL 60611, USA; [email protected] 3 Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60201, USA * Correspondence: [email protected] Received: 30 June 2020; Accepted: 27 July 2020; Published: 29 July 2020 Abstract: The HECT (Homologous to the E6-AP Carboxyl Terminus)-family protein E6AP (E6-associated protein), encoded by the UBE3A gene, is a multifaceted ubiquitin ligase that controls diverse signaling pathways involved in cancer and neurological disorders. The oncogenic role of E6AP in papillomavirus-induced cancers is well known, with its action to trigger p53 degradation in complex with the E6 viral oncoprotein. However, the roles of E6AP in non-viral cancers remain poorly defined. It is well established that loss-of-function alterations of the UBE3A gene cause Angelman syndrome, a severe neurodevelopmental disorder with autosomal dominant inheritance modified by genomic imprinting on chromosome 15q. Moreover, excess dosage of the UBE3A gene markedly increases the penetrance of autism spectrum disorders, suggesting that the expression level of UBE3A must be regulated tightly within a physiologically tolerated range during brain development. In this review, current the knowledge about the substrates of E6AP-mediated ubiquitination and their functions in cancer and neurological disorders is discussed, alongside with the ongoing efforts to pharmacologically modulate this ubiquitin ligase as a promising therapeutic target. Keywords: ubiquitin; viral oncogenesis; oncoproteins; tumor suppressors; Angelman syndrome; autism; small molecules; imprinting; E3 ligase; chromosome 15q 1. Introduction and E6AP Structure Ubiquitination is a major posttranslational modification which governs the fates of modified protein substrates, i.e., changes in stability, subcellular localization and enzymatic activities [1]. The multi-step process of ubiquitination involves the activation, conjugation and ligation of a ubiquitin moiety mediated by E1, E2, and E3 enzymes, respectively. E3 ligases play crucial roles in recruiting substrates and they primarily determine substrate specificity [1]. Aberrant regulation of E3 ligases disrupts diverse cellular functions such as cell cycle control, DNA damage response and cell death, and could shift the balance towards oncogenesis [2]. The human genome encodes more than 600 E3 ligases, which are classified into a few major families based on their structural similarities: RING (Really Interesting New Gene), RBR (ring between ring), Ubox and HECT E3 ligases [3]. In this review, we focus on one of the HECT E3 ligases, E6AP, which is also known as UBE3A and plays roles in oncogenesis, neurodevelopmental disorders, and other human diseases [4]. HECT-E3s ubiquitinate their specific substrate in a two-step process. First, an HECT-E3 binds to an E2 in complex with activated ubiquitin, leading to the formation of a thioester linkage between the C-terminus of ubiquitin and the catalytic cysteine residue in the HECT domain. This transient complex subsequently transfers ubiquitin to an interacting substrate with the formation of an isopeptide bond [5]. Cancers 2020, 12, 2108; doi:10.3390/cancers12082108 www.mdpi.com/journal/cancers Cancers 2020, 12, 2108 2 of 19 Cancers 2020, 12, x 2 of 20 Structurally, E6AP possesses a Zn2+-binding N-terminal (Amino-terminal Zn-finger of Ube3a Ligase (AZUL))(AZUL)) domain domain and and a a catalytic catalytic HECT HECT domain domain of of ~350 ~350 amino amino acids acids at atthe the C Cterminus terminus (Figure (Figure 1a)1a) [6]. [ 6 A]. domainA domain necessary necessary for binding for binding with withthe human the human papillo papillomavirusmavirus (HPV) (HPV)E6 oncoprotein E6 oncoprotein is located is between located thebetween AZUL the and AZUL HECT and domains HECT domains [7]. The[ 7AZUL]. The AZULdomain domain is involved is involved in substrate in substrate recruitment recruitment and andalso self-inhibitoryalso self-inhibitory regulation regulation [8]. [Most8]. Most mutationsmutations associated associated with with the the neurodevelopmental neurodevelopmental disorderdisorder AngelmanAngelman syndrome syndrome (AS) are found in the HECT domain, indicating thatthat the loss of catalytic function ofof E6AP E6AP causes AS [[9].9]. ConsideringConsidering thethe humanhuman E6AP-UBCH7E6AP-UBCH7 complexcomplex crystalcrystal structurestructure (1C4Z.pdb)(1C4Z.pdb) [[6],6], wewe have have extracted extracted out out the the E6AP E6AP part part by by the the Biopolymer Biopolymer module module implemented implemented in Tripos in Tripos software software and highlightedand highlighted AS-associated AS-associated mutations mutations and the andcatalytic the catalytic Cys820 residue Cys820 (Figure residue 1b). (Figure The N1b). lobe The (labeled N lobe in magenta)(labeled inbinds magenta) to the specific binds E2 to partner, the specific UBE2L3/Ubc E2 partner,H7. Many UBE2L3 AS mutation/UbcH7. sites Many are AS located mutation in the areas sites onare both located the C inlobe the (labeled areas onin green) both theand CN lobe su (labeledrrounding in green)Cys820, and while N some lobe mutations surrounding are found Cys820, in thewhile E2-binding some mutations region ofare the foundN lobe, in e.g., the Thr656 E2-binding and Phe690 region [10]. of the AS-lin N lobe,ked e.g.,missense Thr656 mutations and Phe690 have [also10]. beenAS-linked reported missense in the mutations AZUL domain have also[9,11], been suggesting reported inthe the significance AZUL domain of the [9 ,domain11], suggesting in catalytic the regulation.significance Aside of the from domain its ubiquitin in catalytic ligase regulation. function, Aside E6AP from also itsacts ubiquitin as a transcriptional ligase function, co-activator E6AP also of steroidacts as ahormone transcriptional receptors co-activator such as estrogen, of steroid progesterone hormone receptors and androgen such as estrogen,receptors progesterone(ER, PR and andAR, respectively)androgen receptors [4,12,13]. (ER, E6AP PR andcontains AR, respectively) a nuclear localizat [4,12,13ion]. E6APsignal contains that allows a nuclear it to be localization localized signalto the nucleus,that allows and itthree to be LxxL localized motifs, to which the nucleus, are important and three for receptor LxxL motifs, interaction which [4,14,15]. are important The transactivating for receptor functioninteraction of E6AP [4,14, 15has]. Thebeen transactivating mapped to a region function outside of E6AP the HECT has been domain mapped (residues to a region 170–680) outside [4]. the HECTRecent domain evidence (residues suggests 170–680) that [ 4E6AP]. exists in two conformational states of high and low activity. WhileRecent the potent evidence activation suggests of E6AP that by E6AP E6 binding exists is in well two known, conformational the binding states with of HERC2, high and another low HECTactivity. E3, While also stimulates the potent the activation activity of of E6AP E6AP (Figure by E6 1a) binding [16,17]. is Mutations well known, in HERC2 the binding which cause with degradationHERC2, another of HERC2 HECT have E3, been also associated stimulates with the activity an AS-like of E6APsyndrome. (Figure Furt1a)hermore, [ 16,17]. protein Mutations kinase in A-mediatedHERC2 which phosphorylation cause degradation of Thr485, of HERC2 which is have located been outside associated the HECT with domain, an AS-like inhibits syndrome. E6AP activity.Furthermore, An autism-linked protein kinase missense A-mediated mutation phosphorylation that disrupts of this Thr485, phosphorylation which is located site outside causes theenhanced HECT E6APdomain, activity inhibits [18]. E6AP activity. An autism-linked missense mutation that disrupts this phosphorylation site causes enhanced E6AP activity [18]. Figure 1. Cont. Cancers 2020, 12, 2108 3 of 19 Cancers 2020, 12, x 3 of 20 FigureFigure 1. E6AP1. E6AP domains domains and and their their therapeutic therapeutic significance. significance. (a) Domains(a) Domains in the in the primary primary sequence sequence of of E6APE6AP protein. protein. The The numbers numbers in parenthesesin parentheses indicate indicate the the corresponding corresponding amino amino acid acid residues residues of E6AP of E6AP isoformisoform 1 protein.1 protein. The The C-terminus C-terminus of E6APof E6AP protein protein has has the the prototype prototype Homologous Homologous to theto the E6-AP E6-AP CarboxylCarboxyl Terminus Terminus (HECT) (HECT) domain. domain. This This domain domain has ha thes the E2-binding E2-binding site site and and the the catalytic catalytic center center Cys820.Cys820. E6-binding E6-binding domain domain binds binds the HPV the E6HPV oncoprotein. E6 oncoprotein. The Zn-binding The Zn-bindi Amino-terminalng Amino-terminal Zn-finger Zn- of Ube3afinger Ligaseof Ube3a (AZUL) Ligase domain (AZUL) is presentdomain atis thepresen N terminus.t at the N The terminus. HERC2-binding The HERC2-binding domain is required domain is forrequired the association for the with association the partner with HECT the partner E3 HERC2. HECT Small E3 moleculeHERC2. Small inhibitors molecule targeting inhibitors the E6AP-E6 targeting