Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases play a critical role in various cellular pathways, including but not limited to protein trafficking, subcellular localization, innate immune response, viral infections, DNA damage responses and apoptosis. To date, 28 HECT E3 ubiquitin ligases have been identified in humans, and recent studies have begun to reveal how these enzymes control various cellular pathways by catalyzing the post-translational attachment of ubiquitin to their respective substrates. New studies have identified substrates and/or interactors with different members of the HECT E3 ubiquitin ligase family, particularly for E6AP and members of the neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) family. However, there still remains many unanswered questions about the specific roles that each of the HECT E3 ubiquitin ligases have in maintaining cellular homeostasis. The present Review discusses our current understanding on the biological roles of the HECT E3 ubiquitin ligases in the cell and how they contribute to disease development. Expanded investigations on the molecular basis for how and why the HECT E3 ubiquitin ligases recognize and regulate their intracellular substrates will help to clarify the biochemical mechanisms employed by these important enzymes in ubiquitin biology.

Ankyrin repeat (AR) domains are considered the most abundant repeat motif found in eukaryotic proteins. AR domains are predominantly known to mediate specific protein–protein interactions (PPIs) without necessarily recognizing specific primary sequences, nor requiring strict conformity within its own primary sequence. This promiscuity allows for one AR domain to recognize and bind to a variety of intracellular substrates, suggesting that AR-containing proteins may be involved in a wide array of functions. Many AR-containing proteins serve a critical role in biological processes including the ubiquitylation signaling pathway (USP). There is also strong evidence that AR-containing protein malfunction are associated with several neurological diseases and disorders. In this review, the structure and mechanism of key AR-containing proteins are discussed to suggest and/or identify how each protein utilizes their AR domains to support ubiquitylation and the cascading pathways that follow upon substrate modification.

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Gankyrin is an oncoprotein responsible for the development of numerous cancer types. It regulates the expression levels of multiple tumor suppressor proteins (TSPs) in liver cancer; however, gankyrin’s regulation of these TSPs in breast and lung cancers has not been thoroughly investigated. Additionally, no small-molecule gankyrin inhibitor has been developed which demonstrates potent anti-proliferative activity against gankyrin overexpressing breast and lung cancers. Herein, we are reporting the structure-based design of gankyrin-binding small molecules which potently inhibited the proliferation of gankyrin overexpressing A549 and MDA-MB-231 cancer cells, reduced colony formation, and inhibited the growth of 3D spheroids in an in vitro tumor simulation model. Investigations demonstrated that gankyrin inhibition occurs through either stabilization or destabilization of its 3D structure. These studies shed light on the mechanism of small-molecule inhibition of gankyrin and demonstrate that gankyrin is a viable therapeutic target for the treatment of breast and lung cancer.

HECT E3 ubiquitin ligases selectively recognize, bind, and ubiquitylate their substrate proteins to target them for 26S proteasomal degradation. There is increasing evidence that HECT E3 ubiquitin ligase dysfunction due to misfolding and/or the gene encoding the protein being mutated is responsible for the development of different diseases. Apart from the more prominent and well-characterized E6AP and members of the NEDD4 family, new studies have begun to reveal how other members of the HECT E3 ubiquitin ligase family function as well as their links to disease and developmental disorders. This chapter provides a comprehensive discussion on the more mysterious members of the HECT E3 ubiquitin ligase family and how they control intracellular processes. Specifically, AREL1, HACE1, HECTD1, HECTD4, G2E3, and TRIP12 will be examined as these enzymes have recently been identified as contributors to disease development.2 covalently attach ubiquitin on to a lysine residue of the substrate protein forming a stable isopeptide bond between the C-terminus of ubiquitin and the ε-amine of the substrate lysine [3,5,6]. This process can be repeated numerous times to form different polyubiquitin chain linkages with the specific HECT E3 ubiquitin ligase dictating the type(s) of ubiquitin linkages that are built [2,7]. Chain typesLinker Proposed function Monoubiquitylation Monoubiquitylation/ multi-monoubiquitylation Endocytosis [9] DNA damage repair [10-15] Histone regulation [10-15] Mitophagy [10-15] Protein localization [10-15] Protein interactions [10-15] Protein transportation [10-15] Transcription activation [10-15] Polyubiquitylation Chain (homotypic) M1 Innate immunity [2, 9, 16] Linear chain formation [9] NF-κB activation [9, 16] Signaling cascades [9, 16] K6 DNA damage response [14] NF-κB regulation [14] Mitophagy [14] K11 Cell cycle regulation [17] DNA damage response [18] Mitophagy [17] NF-κB activation [16] Protein degradation [17] K27 DNA damage response [18] Kinase activation [19] Protein degradation [20] Protein scaffolding [21] Protein trafficking [22] K29 DNA damage response [18] Kinase activation [19] Protein degradation [9] K33 DNA damage response [10-15, 18] Kinase activation [23] Post-golgi trafficking [24] T-cell signaling [23] K4 8 Protein degradation [1, 2, 25] K63 DNA damage response [9, 18] NF-κB activation [9, 16] Protein trafficking [9] Chain (heterotypic; branched) M1 /K63 NF-κB activation [16] K11 /K4 8 Protein degradation [26, 27] K29/K48 Protein degradation [26] K48/K63 Protein degradation [26] K11 /K63 Endocytosis [28]