Ubiquitin chain specificities of E6AP E3 ligase and its HECT domain

Abstract Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

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Conformational Flexibility Underlies Ubiquitin Ligation Mediated by the WWP1 HECT Domain E3 Ligase

Molecular Cell ◽

10.1016/s1097-2765(02)00774-8 ◽

2003 ◽

Vol 11 (1) ◽

pp. 249-259 ◽

Cited By ~ 180

Author(s):

Mark A Verdecia ◽

Claudio A.P Joazeiro ◽

Nicholas J Wells ◽

Jean-Luc Ferrer ◽

Marianne E Bowman ◽

...

Keyword(s):

E3 Ligase ◽

Conformational Flexibility ◽

Hect Domain

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Novel Approach for Characterizing Ubiquitin E3 Ligase Function

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110380456 ◽

2010 ◽

Vol 15 (10) ◽

pp. 1220-1228 ◽

Cited By ~ 15

Author(s):

Jeffrey G. Marblestone ◽

K. G. Suresh Kumar ◽

Michael J. Eddins ◽

Craig A. Leach ◽

David E. Sterner ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput Screening ◽

E3 Ligase ◽

Cost Effective ◽

Ubiquitin Proteasome System ◽

Dependent Manner ◽

Ubiquitin Chain ◽

E3 Ligases ◽

Effective Manner ◽

Wide Range

The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)–compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform’s broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.

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Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Biochemical Journal ◽

10.1042/bcj20200027 ◽

2020 ◽

Vol 477 (5) ◽

pp. 905-923 ◽

Cited By ~ 1

Author(s):

Sunil Singh ◽

J. Sivaraman

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Enzymatic Activity ◽

Cancer Metastasis ◽

E3 Ligase ◽

The Other ◽

Hect Domain ◽

Loop Region ◽

The Stability

The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

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1,4-Naphthoquinones as inhibitors of Itch, a HECT domain-E3 ligase, and tumor growth suppressors in multiple myeloma

European Journal of Medicinal Chemistry ◽

10.1016/j.ejmech.2017.09.011 ◽

2017 ◽

Vol 140 ◽

pp. 84-91 ◽

Cited By ~ 5

Author(s):

Yi-Min Liu ◽

Wei-Chun HuangFu ◽

Han-Li Huang ◽

Wei-Cheng Wu ◽

Yi-Lin Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

E3 Ligase ◽

Hect Domain

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E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1017516108 ◽

2011 ◽

Vol 108 (14) ◽

pp. 5590-5595 ◽

Cited By ~ 76

Author(s):

R. G. Hibbert ◽

A. Huang ◽

R. Boelens ◽

T. K. Sixma

Keyword(s):

E3 Ligase ◽

Ubiquitin Chain ◽

Chain Formation

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Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

10.1101/2021.11.25.470069 ◽

2021 ◽

Author(s):

Lu Zhu ◽

Qing Zhang ◽

Ciro Cordeiro ◽

Sudeep Banjade ◽

Richa Sardana ◽

...

Keyword(s):

E3 Ligase ◽

Adaptor Proteins ◽

Regulation Mechanism ◽

Hect Domain ◽

E3 Ligases ◽

Cellular Processes ◽

Protein Ubiquitination ◽

Cargo Protein ◽

Py Motif ◽

Substrate Induction

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

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Corrigendum to “1,4-Naphthoquinones as inhibitors of Itch, a HECT domain-E3 ligase, and tumor growth suppressors in multiple myeloma” [Eur. J. Med. Chem. 140 (2017) 84–91]

European Journal of Medicinal Chemistry ◽

10.1016/j.ejmech.2020.112633 ◽

2020 ◽

Vol 202 ◽

pp. 112633

Author(s):

Yi-Min Liu ◽

Wei-Chun HuangFu ◽

Han-Li Huang ◽

Wei-Cheng Wu ◽

Yi-Lin Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

E3 Ligase ◽

Hect Domain

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Linear ubiquitin chains: enzymes, mechanisms and biology

Open Biology ◽

10.1098/rsob.170026 ◽

2017 ◽

Vol 7 (4) ◽

pp. 170026 ◽

Cited By ~ 48

Author(s):

Katrin Rittinger ◽

Fumiyo Ikeda

Keyword(s):

Cellular Response ◽

Apoptotic Cell ◽

Gene Mutations ◽

E3 Ligase ◽

Biological Functions ◽

Post Translational Modification ◽

Ubiquitin Chain ◽

Polyubiquitin Chains ◽

Ubiquitin E3 Ligase ◽

Ubiquitin Receptors

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases.

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