Photocontrollable PROTAC molecules: Structure and mechanism of action

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

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Ubiquitination, Biotech Startups, and the Future of TRIM Family Proteins: A TRIM-Endous Opportunity

Cells ◽

10.3390/cells10051015 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1015

Author(s):

Utsa Bhaduri ◽

Giuseppe Merla

Keyword(s):

Drug Discovery ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Genetic Disorders ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

E3 Ubiquitin Ligases ◽

Post Translational Modification ◽

Disease Biology ◽

The Future

Ubiquitination is a post-translational modification that has pivotal roles in protein degradation and diversified cellular processes, and for more than two decades it has been a subject of interest in the biotech or biopharmaceutical industry. Tripartite motif (TRIM) family proteins are known to have proven E3 ubiquitin ligase activities and are involved in a multitude of cellular and physiological events and pathophysiological conditions ranging from cancers to rare genetic disorders. Although in recent years many kinds of E3 ubiquitin ligases have emerged as the preferred choices of big pharma and biotech startups in the context of protein degradation and disease biology, from a surface overview it appears that TRIM E3 ubiquitin ligases are not very well recognized yet in the realm of drug discovery. This article will review some of the blockbuster scientific discoveries and technological innovations from the world of ubiquitination and E3 ubiquitin ligases that have impacted the biopharma community, from biotech colossuses to startups, and will attempt to evaluate the future of TRIM family proteins in the province of E3 ubiquitin ligase-based drug discovery.

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00477-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Zhou Yu ◽

Xuelian Li ◽

Mingjin Yang ◽

Jiaying Huang ◽

Qian Fang ◽

...

Keyword(s):

Nucleic Acids ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

B Cell Lymphoma ◽

Antiviral Response ◽

Type I Interferons ◽

Type I ◽

Innate Response ◽

Innate Antiviral Response

AbstractSensing of pathogenic nucleic acids by pattern recognition receptors (PRR) not only initiates anti-microbe defense but causes inflammatory and autoimmune diseases. E3 ubiquitin ligase(s) critical in innate response need to be further identified. Here we report that the tripartite motif-containing E3 ubiquitin ligase TRIM41 is required to innate antiviral response through facilitating pathogenic nucleic acids-triggered signaling pathway. TRIM41 deficiency impairs the production of inflammatory cytokines and type I interferons in macrophages after transfection with nucleic acid-mimics and infection with both DNA and RNA viruses. In vivo, TRIM41 deficiency leads to impaired innate response against viruses. Mechanistically, TRIM41 directly interacts with BCL10 (B cell lymphoma 10), a core component of CARD proteins−BCL10 − MALT1 (CBM) complex, and modifies the Lys63-linked polyubiquitylation of BCL10, which, in turn, hubs NEMO for activation of NF-κB and TANK-binding kinase 1 (TBK1) − interferon regulatory factor 3 (IRF3) pathways. Our study suggests that TRIM41 is the potential universal E3 ubiquitin ligase responsible for Lys63 linkage of BCL10 during innate antiviral response, adding new insight into the molecular mechanism for the control of innate antiviral response.

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p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLprovia E3 ubiquitin ligase RCHY1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603435113 ◽

2016 ◽

Vol 113 (35) ◽

pp. E5192-E5201 ◽

Cited By ~ 55

Author(s):

Yue Ma-Lauer ◽

Javier Carbajo-Lozoya ◽

Marco Y. Hein ◽

Marcel A. Müller ◽

Wen Deng ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Nonstructural Protein ◽

E3 Ubiquitin Ligase ◽

Ring Finger ◽

Immune Recognition ◽

Major Player ◽

Nonstructural Protein 3 ◽

Human Coronaviruses ◽

Unique Domain

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLprofusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLproalone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

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Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

Open Biology ◽

10.1098/rsob.130172 ◽

2014 ◽

Vol 4 (3) ◽

pp. 130172 ◽

Cited By ~ 13

Author(s):

Barbara Franke ◽

Alexander Gasch ◽

Dayté Rodriguez ◽

Mohamed Chami ◽

Muzamil M. Khan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Quaternary Structure ◽

E3 Ubiquitin Ligase ◽

Coiled Coil ◽

Trim Protein ◽

Muscle Catabolism ◽

And Function ◽

Helical Region ◽

Structural Significance

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

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Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e12-07-0548 ◽

2012 ◽

Vol 23 (21) ◽

pp. 4203-4211 ◽

Cited By ~ 9

Author(s):

Dong-Hwan Kim ◽

Deanna M. Koepp

Keyword(s):

Cell Cycle ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

S Phase ◽

Temperature Sensitive ◽

Dependent Manner ◽

Charged Residues ◽

Phase Progression ◽

Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽

10.1182/blood.2020006528 ◽

2021 ◽

Author(s):

Roger Belizaire ◽

Sebastian Hassan John Koochaki ◽

Namrata D. Udeshi ◽

Alexis Vedder ◽

Lei Sun ◽

...

Keyword(s):

Cell Lines ◽

Ubiquitin Ligase ◽

Therapeutic Potential ◽

Mutant Cell ◽

E3 Ubiquitin Ligase ◽

Akt Signaling ◽

Allelic Series ◽

Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

Cell Death and Differentiation ◽

10.1038/s41418-019-0406-6 ◽

2019 ◽

Vol 27 (4) ◽

pp. 1200-1213 ◽

Cited By ~ 1

Author(s):

Ainhoa Nieto ◽

Makoto R. Hara ◽

Victor Quereda ◽

Wayne Grant ◽

Vanessa Saunders ◽

...

Keyword(s):

Dna Damage ◽

Ionizing Radiation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Molecular Scaffold ◽

Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

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