Targeting <scp>HECT</scp>‐type E3 ligases – insights from catalysis, regulation and inhibitors

Fajner, Valentina; Maspero, Elena; Polo, Simona

doi:10.1002/1873-3468.12775

Cited by 61 publications

(46 citation statements)

References 82 publications

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“…E3 ubiquitin ligases play a crucial role in this cascade, which affects both the efficiency and substrate specificity (Zheng & Shabek, 2017). E3 ligases are divided into three classes according to conserved functional and structural features: really interesting new gene, homologous to E6-AP carboxyl terminus (HECT), and ring between ring (Fajner, Maspero, & Polo, 2017). HECT E3s are defined by a 350 residues module, which is first characterised in the human E3 ligase E6-associated protein E6AP positioned invariably at the C-terminal (Huibregtse, Scheffner, Beaudenon, & Howley, 1995).…”

Section: Introductionmentioning

confidence: 99%

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The serine/threonine protein kinase ofStreptococcus suisserotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier

Liu

Meng

et al. 2018

Cellular Microbiology

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Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB-1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified and was found to contain a TnYLB-1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood-brain barrier (BBB). We observed that the SS2 wild-type ZY05719 strain crossed the BBB model more readily than the mutant strain. Hence, we speculated that STK is associated with the ability of crossing blood-brain barrier in SS2. In vitro, compared with ZY05719, the ability of the stk-deficient strain (Δstk) to adhere to and invade both hBMECs and bEnd.3 cells, as well as to cross the BBB, was significantly attenuated. Immunocytochemistry using antibodies against claudin-5 in bEnd.3 cells showed that infection by ZY05719 disrupted BBB tight junction proteins to a greater extent than in infection by Δstk. The studies revealed that SS2 initially binds at or near intercellular junctions and crosses the BBB via paracellular traversal. Claudin-5 mRNA levels were indistinguishable in ZY05719- and Δstk-infected cells. This result indicated that the decrease of claudin-5 was maybe induced by protein degradation. Cells infected by ZY05719 exhibited higher ubiquitination levels than cells infected by Δstk. This result indicated that ubiquitination was involved in the degradation of claudin-5. Differential proteomic analysis showed that E3 ubiquitin protein ligase HECTD1 decreased by 1.5-fold in Δstk-infected bEnd.3 cells relative to ZY05719-infected cells. Together, the results suggested that STK may affect the expression of E3 ubiquitin ligase HECTD1 and subsequently increase the degradation of claudin-5, thus enabling SS2 to traverse the BBB.

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Section: Introductionmentioning

confidence: 99%

“…E3 ubiquitin ligase HECTD1 is one of the E3 ligases, which contains ankyrin repeats, UBE3B, and UBE3C, which carry an IQ domain (Fajner et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

The serine/threonine protein kinase ofStreptococcus suisserotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier

Liu

Meng

et al. 2018

Cellular Microbiology

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“…The E3 ligase for Rpn‐13—Ube3C—is already known. Ube3C has limited tertiary structure to which a small‐molecule compound might bind, so it is currently unknown whether this E3 ligase could be used in the PROTAC mechanism of degradation …”

Section: Discussionmentioning

confidence: 99%

Small‐Molecule Inhibitors of the Proteasome's Regulatory Particle

2019

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Cells need to synthesize and degrade proteins consistently. Maintaining a balanced level of protein in the cell requires a carefully controlled system and significant energy. Degradation of unwanted or damaged proteins into smaller peptide units can be accomplished by the proteasome. The proteasome is composed of two main subunits. The first is the core particle (20S CP), and within this core particle are three types of threonine proteases. The second is the regulatory complex (19S RP), which has a myriad of activities including recognizing proteins marked for degradation and shuttling the protein into the 20S CP to be degraded. Small‐molecule inhibitors of the 20S CP have been developed and are exceptional treatments for multiple myeloma (MM). 20S CP inhibitors disrupt the protein balance, leading to cellular stress and eventually to cell death. Unfortunately, the 20S CP inhibitors currently available have dose‐limiting off‐target effects and resistance can be acquired rapidly. Herein, we discuss small molecules that have been discovered to interact with the 19S RP subunit or with a protein closely associated with 19S RP activity. These molecules still elicit their toxicity by preventing the proteasome from degrading proteins, but do so through different mechanisms of action.

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“…E3 enzymes achieve ubiquitylation by recruiting one or more sequence motifs, termed "degrons", in a substrate, and promoting Ub transfer through one of various mechanisms determined by the type of catalytic domain. E3s harboring "HECT" and "RBR" catalytic domains promote ubiquitylation through 2-step reactions involving formation of a thioester-linked intermediate between the E3 and Ub's C-terminus: first, Ub is transferred from an E2~Ub intermediate ("~" refers to thioester bond) to the E3 catalytic Cys; Ub is subsequently transferred from the E3's Cys to the substrate (9)(10)(11)(12)(13). Alternatively, the majority of E3s, including an estimated ≈500 RING E3s in humans, do not directly relay Ub themselves, but instead activate Ub transfer from the catalytic Cys of a Ub-carrying enzyme, which is typically an E2, but can also be a thioester-forming E3 (14,15).…”

Section: Introductionmentioning

confidence: 99%

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Watson

Grace

Zhang

et al. 2019

Preprint

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Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin's C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at "exosites". However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.

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Targeting HECT‐type E3 ligases – insights from catalysis, regulation and inhibitors

Cited by 61 publications

References 82 publications

The serine/threonine protein kinase ofStreptococcus suisserotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier

The serine/threonine protein kinase ofStreptococcus suisserotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier

Small‐Molecule Inhibitors of the Proteasome's Regulatory Particle

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

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