Homologous To E6AP C Terminus Domain Research Articles

The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5.

Ubiquitin (Ub) chain formation by homologous to E6AP C-terminus (HECT)-family E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We used chemistry and cryo-electron microscopy (cryo-EM) to visualize stable mimics of the intermediates along K48-linked Ub chain formation by the human E3, UBR5. The structural data reveal a ≈ 620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly tethered Ub-associated (UBA) domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor Ub, with its K48 lured into the active site by numerous interactions between the acceptor Ub, manifold UBR5 elements and the donor Ub. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing Ub transfer from E2 to E3 and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2 and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked Ub chain forging machine.

The Role of HECT E3 Ubiquitin Ligases in Colorectal Cancer.

Colorectal cancer (CRC) is estimated to rank as the second reason for cancer-related deaths, and the prognosis of CRC patients remains unsatisfactory. Numerous studies on gastrointestinal cell biology have shown that the E3 ligase-mediated ubiquitination exerts key functions in the pathogenesis of CRC. The homologous to E6-associated protein C-terminus (HECT) family E3 ligases are a major group of E3 enzymes, featured with the presence of a catalytic HECT domain, which participate in multiple cellular processes; thus, alterations in HECT E3 ligases in function or expression are closely related to the occurrence and development of many human malignancies, including-but not limited to-CRC. In this review, we summarize the potential role of HECT E3 ligases in colorectal carcinogenesis and the related underlying molecular mechanism to expand our understanding of their pathological functions. Exploiting specific inhibitors targeting HECT E3 ligases could be a potential therapeutic strategy for CRC therapy in the future.

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family.

There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.

Extending the Catalytic HECT Domain Boundaries for the HECT E3 Ubiquitin Ligase HACE1 Increases Solubility and Enzymatic Activity

There are 28 unique members of the homologous to E6AP C‐terminus (HECT) E3 ubiquitin ligase family responsible for catalytically transferring a ubiquitin moiety onto a specific substrate. This family contains a relatively conserved C­‐terminal HECT domain that mediates its ubiquitylation activity with a highly conserved cysteine residue. Of these, HECT Domain and Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1 (HACE1) is one of the ‘other’ HECT E3 ubiquitin ligases containing an N‐terminal ankyrin repeat domain responsible for facilitating substrate recognition and binding. Isolation of a biochemically pure HECT domain has proven difficult due to chronic insolubility and/or inhibited enzymatic activity. Recent in‐silico bioinformatic analyses coupled with secondary structure prediction software indicated a conserved amphipathic α‐helix preceding the N‐termini in all HECT E3 ubiquitin ligases. This α ‐helix is proposed to bind to a hydrophobic pocket found within the HECT domain. Building on recently published structural studies and in‐house biochemical and biophysical experiments, we show a drastic increase in protein yield, solubility, stability, and activity of the HECT domain of HACE1. This finding also holds true for other members of the HECT E3 ubiquitin ligase family. Our cumulative findings support a call to redefine the boundaries of the HECT domain to include this N‐terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.

Overexpressed Smurf1 is degraded in glioblastoma cells through autophagy in a p62-dependent manner.

Homologous to E6AP C‐terminus (HECT)‐type E3 ubiquitin ligase SMAD‐specific E3 ubiquitin protein ligase 1 (Smurf1) was originally identified to ubiquitinate Smad protein in the TGF‐β/BMP signaling pathway. Recently, Smurf1 has been reported to promote tumorigenesis by regulating multiple biological processes. High expression of Smurf1 plays a vital role in brain tumor progression by mediating aberrant cell signaling pathways. Previous reports have shown that Smurf1 is degraded mainly through the ubiquitin–proteasome system, but it remains unclear whether Smurf1 is degraded by autophagy in tumor cells. In this study, we show that autophagy activators promote Smurf1 degradation in glioblastoma (GB) cells. The autophagy receptor p62 colocalizes with ubiquitinated substrates to promote sequestration of cytoplasm cargo into the autophagosome. We report that autophagic degradation of Smurf1 is dependent on p62. Moreover, the autophagic degradation of Smurf1 is prevented in the absence of the HECT domain or E3 ubiquitin ligase activity. We further proved that activation of autophagy leads to a decrease of Smurf1 and the inhibition of the phosphoinositide 3‐kinase/protein kinase B signaling pathway in GB cells. Our results suggest that enhancement of autophagic degradation of Smurf1 may be a potential approach to treating GB.

UBE3A Enzyme Hyperactivity as a Driver of Neurodevelopmental Disease

UBE3A is a HECT (homologous to E6AP C-terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin-proteasome pathway. Deletion or null mutation of the maternal UBE3A gene causes Angelman syndrome, a severe intellectual disability disorder also characterized by epilepsy, motor deficits, and an unusually happy disposition. Hundreds of single amino acid variants of UBE3A have been reported in the literature, but the biological significance for most these variants are unknown. Here, we devised a high-throughput assay to screen the functional consequence of UBE3A missense variants to identify precise mutations that drive disease. We screened over 150 UBE3A variants and identified distinct functional classes of UBE3A mutants, including over a dozen gain-of-function variants previously not known to exist. Mice carrying a specific hyperactivating mutation in UBE3A exhibited aberrant motor and communication deficits, and analysis of patient data showed that UBE3A hyperactivation causes a neurodevelopmental disorder that is distinct from Angelman syndrome. Finally, our detailed structure-function analysis revealed a novel allosteric regulatory site in UBE3A that is perturbed by both gain and loss-of-function mutations. Altogether, our study demonstrates that functional variant analysis can delineate disease subtypes and provide deep structure-functional data to uncover disease-relevant regulatory mechanisms.

Adaptors as the regulators of HECT ubiquitin ligases.

The HECT (homologous to E6AP C-terminus) ubiquitin ligases (E3s) are a small family of highly conserved enzymes involved in diverse cellular functions and pathological conditions. Characterised by a C-terminal HECT domain that accepts ubiquitin from E2 ubiquitin conjugating enzymes, these E3s regulate key signalling pathways. The activity and functional regulation of HECT E3s are controlled by several factors including post-translational modifications, inter- and intramolecular interactions and binding of co-activators and adaptor proteins. In this review, we focus on the regulation of HECT E3s by accessory proteins or adaptors and discuss various ways by which adaptors mediate their regulatory roles to affect physiological outcomes. We discuss common features that are conserved from yeast to mammals, regardless of the type of E3s as well as shed light on recent discoveries explaining some existing enigmas in the field.

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.

Structure and Function of HECT E3 Ubiquitin Ligases and their Role in Oxidative Stress.

Ubiquitination is a modification after protein transcription that plays a vital role in maintaining the homeostasis of the cellular environment. The Homologous to E6AP C-terminus (HECT) family E3 ubiquitin ligases are a kind of E3 ubiquitin ligases with a C-terminal HECT domain that mediates the binding of ubiquitin to substrate proteins and a variable-length N-terminal extension. HECT-ubiquitinated ligases can be divided into three categories: NEDD4 superfamily, HERC superfamily, and other HECT superfamilies. HECT ubiquitin ligase plays an essential role in the development of many human diseases. In this review, we focus on the physiological and pathological processes involved in oxidative stress and the role of E3 ubiquitin ligase of the HECT family.

A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5.

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 invivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are "unlocked" by Ubp2-mediated deubiquitination.

The E3 ligase Itch in immune regulation and beyond.

Itch or itchy E3 ubiquitin ligase was initially discovered by genetic studies on the mouse coat color changes, and its deletion results in an itchy phenotype with constant skin scratching and multi-organ inflammation. It is a member of the homologous to E6-associated protein C-terminus (HECT)-type family of E3 ligases, with the protein-interacting WW-domains for the recruitment of substrate and the HECT domain for the transfer of ubiquitin to the substrate. Since its discovery, numerous studies have demonstrated that Itch is involved in the control of many aspects of immune responses including T-cell activation and tolerance and T-helper cell differentiation. Itch is also implicated in other biological contexts such as tumorigenesis, development, and stress responses. Many signaling pathways are regulated by Itch-promoted ubiquitylation of diverse target proteins. Itch is also involved in human diseases. Here, we discuss the major progress in understanding the biological significance of Itch-promoted protein ubiquitylation in the immune and other systems and in Itch-mediated regulation of signal transduction.

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases.

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.

Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming

Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT(Nedd4)~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue.

Structure of the HECT C-lobe of the UBR5 E3 ubiquitin ligase.

UBR5 ubiquitin ligase (also known as EDD, Rat100 or hHYD) is a member of the E3 protein family of HECT (homologous to E6-AP C-terminus) ligases as it contains a C-terminal HECT domain. In ubiquitination cascades involving E3s of the HECT class, ubiquitin is transferred from an associated E2 ubiquitin-conjugating enzyme to the acceptor cysteine of the HECT domain, which consists of structurally distinct N- and C-lobes connected by a flexible linker. Here, the high-resolution crystal structure of the C-lobe of the HECT domain of human UBR5 is presented. The structure reveals important features that are unique compared with other HECT domains. In particular, a distinct four-residue insert in the second helix elongates this helix, resulting in a strikingly different orientation of the preceding loop. This protruding loop is likely to contribute to specificity towards the E2 ubiquitin-conjugating enzyme UBCH4, which is an important functional partner of UBR5. Ubiquitination assays showed that the C-lobe of UBR5 is able to form a thioester-linked E3-ubiquitin complex, although it does not physically interact with UBCH4 in NMR experiments. This study contributes to a better understanding of UBR5 ubiquitination activity.

Novel E3 Ubiquitin Ligases That Regulate Histone Protein Levels in the Budding Yeast Saccharomyces cerevisiae

Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.

SCFFBXL15regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.

The ubiquitin ligase E6‐AP promotes degradation of α‐synuclein

Parkinson's disease (PD) is a common neurodegenerative disorder caused mainly because of the loss of dopaminergic neurons in the substantia nigra. Protein inclusions called Lewy bodies are the most common pathological hallmark of PD and other synucleinopathies. Because the main component of these inclusions is alpha-synuclein, aggregation of this protein is thought to be a key pathogenic event in this disease. In the present investigation we report that E6 associated protein (E6-AP), a HECT (homologous to E6-AP C-terminus) domain ubiquitin ligase is a component of Lewy bodies in post-mortem PD brain. In the cell culture model, we demonstrate that endogenous E6-AP colocalizes with alpha-synuclein in juxtanuclear aggregates. E6-AP is also recruited to the centrosome upon inhibition of the proteasome function suggesting its involvement in the degradation of misfolded proteins. Over-expression of E6-AP enhances the degradation of wild type as well as the mutant forms of alpha-synuclein in a proteasome-dependent manner. E6-AP also promotes the degradation of the more toxic oligomeric forms of alpha-synuclein. Our data suggests that E6-AP is involved in the clearance of alpha-synuclein.

Regulation of the RSP5 Ubiquitin Ligase by an Intrinsic Ubiquitin-binding Site

Rsp5 is a homologous to E6AP C terminus (HECT) ubiquitin ligase (E3) that controls many different cellular processes in budding yeast. Although Rsp5 targets a number of different substrates for ubiquitination, the mechanisms that regulate Rsp5 activity remain poorly understood. Here we demonstrate that Rsp5 carries a noncovalent ubiquitin-binding site in its catalytic HECT domain. The N-terminal lobe of the HECT domain mediates binding to ubiquitin, and point mutations that disrupt interactions with ubiquitin alter the ability of the Rsp5 HECT domain to assemble polyubiquitin chains in vitro. Point mutations that disrupt ubiquitin binding also result in temperature-sensitive growth defects in yeast, indicating that the Rsp5 ubiquitin-binding site is important for Rsp5 function in vivo. The Nedd4 HECT domain N-lobe also contains ubiquitin-binding activity, suggesting that interactions between the N-lobe and ubiquitin are conserved within the Nedd4 family of ubiquitin ligases. We propose that a subset of HECT E3s are regulated by a conserved ubiquitin-binding site that functions to restrict the length of polyubiquitin chains synthesized by the HECT domain.