Proteolysis-targeting chimeras mediate the degradation of bromodomain and extra-terminal domain proteins

2020 ◽  
Vol 12 (18) ◽  
pp. 1669-1683
Author(s):  
Yifei Yang ◽  
Zhenwei Wu ◽  
Pan Chen ◽  
Peiyuan Zheng ◽  
Huibin Zhang ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Side Effects ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Drug Discovery And Development ◽  
Terminal Domain ◽  
Super Enhancer ◽  
Bet Inhibitors ◽  
Subsequent Degradation ◽  
Bet Protein

Bromodomain and extra-terminal domain (BET) protein family plays an important role in regulating gene transcription preferentially at super-enhancer regions and has been involved with several types of cancers as a candidate. Up to now, there are 16 pan-BET inhibitors in clinical trials, however, most of them have undesirable off-target and side-effects. The proteolysis-targeting chimeras technology through a heterobifunctional molecule to link the target protein and E3 ubiquitin ligase, causes the target’s ubiquitination and subsequent degradation. By using this technology, the heterobifunctional small-molecule BET degraders can induce BET protein degradation. In this review, we discuss the advances in the drug discovery and development of BET-targeting proteolysis-targeting chimeras.

Treatment of Lymphoid and Myeloid Malignancies by Immunomodulatory Drugs

2019 ◽  
Vol 19 (1) ◽  
pp. 51-78 ◽  
Author(s):  
Ota Fuchs
Keyword(s):  
Clinical Trials ◽  
Transcription Factors ◽  
Ubiquitin Ligase ◽  
Mononuclear Cells ◽  
E3 Ubiquitin Ligase ◽  
Lymphocytic Leukemia ◽  
Monocarboxylate Transporter ◽  
Immunomodulatory Drugs ◽  
Monocarboxylate Transporter 1 ◽  
Argonaute 2

Thalidomide and its derivatives (lenalidomide, pomalidomide, avadomide, iberdomide hydrochoride, CC-885 and CC-90009) form the family of immunomodulatory drugs (IMiDs). Lenalidomide (CC5013, Revlimid®) was approved by the US FDA and the EMA for the treatment of multiple myeloma (MM) patients, low or intermediate-1 risk transfusion-dependent myelodysplastic syndrome (MDS) with chromosome 5q deletion [del(5q)] and relapsed and/or refractory mantle cell lymphoma following bortezomib. Lenalidomide has also been studied in clinical trials and has shown promising activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Lenalidomide has anti-inflammatory effects and inhibits angiogenesis. Pomalidomide (CC4047, Imnovid® [EU], Pomalyst® [USA]) was approved for advanced MM insensitive to bortezomib and lenalidomide. Other IMiDs are in phases 1 and 2 of clinical trials. Cereblon (CRBN) seems to have an important role in IMiDs action in both lymphoid and myeloid hematological malignancies. Cereblon acts as the substrate receptor of a cullin-4 really interesting new gene (RING) E3 ubiquitin ligase CRL4CRBN. This E3 ubiquitin ligase in the absence of lenalidomide ubiquitinates CRBN itself and the other components of CRL4CRBN complex. Presence of lenalidomide changes specificity of CRL4CRBN which ubiquitinates two transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos), and casein kinase 1α (CK1α) and marks them for degradation in proteasomes. Both these transcription factors (IKZF1 and IKZF3) stimulate proliferation of MM cells and inhibit T cells. Low CRBN level was connected with insensitivity of MM cells to lenalidomide. Lenalidomide decreases expression of protein argonaute-2, which binds to cereblon. Argonaute-2 seems to be an important drug target against IMiDs resistance in MM cells. Lenalidomide decreases also basigin and monocarboxylate transporter 1 in MM cells. MM cells with low expression of Ikaros, Aiolos and basigin are more sensitive to lenalidomide treatment. The CK1α gene (CSNK1A1) is located on 5q32 in commonly deleted region (CDR) in del(5q) MDS. Inhibition of CK1α sensitizes del(5q) MDS cells to lenalidomide. CK1α mediates also survival of malignant plasma cells in MM. Though, inhibition of CK1α is a potential novel therapy not only in del(5q) MDS but also in MM. High level of full length CRBN mRNA in mononuclear cells of bone marrow and of peripheral blood seems to be necessary for successful therapy of del(5q) MDS with lenalidomide. While transfusion independence (TI) after lenalidomide treatment is more than 60% in MDS patients with del(5q), only 25% TI and substantially shorter duration of response with occurrence of neutropenia and thrombocytopenia were achieved in lower risk MDS patients with normal karyotype treated with lenalidomide. Shortage of the biomarkers for lenalidomide response in these MDS patients is the main problem up to now.

scholarly journals Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics

2017 ◽  
Vol 28 (3) ◽  
pp. 396-410 ◽  
Author(s):  
Edward Cherok ◽  
Shan Xu ◽  
Sunan Li ◽  
Shweta Das ◽  
W. Alex Meltzer ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Mitochondrial Morphology ◽  
Ubiquitinated Proteins ◽  
Terminal Domain ◽  
Degradation Rates ◽  
And Control

MARCH5, an OMM-associated E3 ubiquitin ligase, controls mitochondrial function. Despite its importance, the mechanism and factors controlling MARCH5 activity are largely unknown. Here we report that the MARCH5 C-terminal domain plays a critical role in degradation of MARCH5 substrates, likely by facilitating release of ubiquitinated proteins from the OMM. We also found that the mitochondrial fission proteins Drp1 and Mff negatively regulate MARCH5’s activity toward MiD49 and Mcl1. Knockouts of either Drp1 or Mff led to reduced expression, shorter half-lives, and increased ubiquitination of MiD49 and Mcl1. Effects of Mff and Drp1 depletion on degradation rates and ubiquitination of Mcl1 and MiD49 were eliminated in Drp1−/−/MARCH5−/− and Mff−/−/MARCH5−/− cells. Our data show that it is not mitochondrial morphology per se but rather Mff and Drp1 that directly control MARCH5. Consistently, we find that Mff is an integral component of the MARCH5/p97/Npl4 complex, which is also controlled by MARCH5’s C-terminal domain. Furthermore, not only mitochondrial fission but also fusion is regulated through Mff and Drp1 protein activities. Thus, in addition to their canonical roles in mitochondrial fission, Mff and Drp1 also act as regulatory factors that control mitochondrial fission and fusion.

scholarly journals A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62

2010 ◽  
Vol 30 (13) ◽  
pp. 3275-3285 ◽  
Author(s):  
Alexandria Lau ◽  
Xiao-Jun Wang ◽  
Fei Zhao ◽  
Nicole F. Villeneuve ◽  
Tongde Wu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Defense Mechanism ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Direct Interaction ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Ubiquitin Ligase Complex ◽  
Subsequent Degradation ◽  
Kelch Domain

ABSTRACT In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

scholarly journals BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Bo Zhang ◽  
Mattias Holmlund ◽  
Severine Lorrain ◽  
Mikael Norberg ◽  
László Bakó ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Flower Development ◽  
Red Light ◽  
E3 Ubiquitin Ligase ◽  
Plant Responses ◽  
Signaling Mechanism ◽  
Ubiquitin Ligase Complex ◽  
Molecular Determinants ◽  
Subsequent Degradation

Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the BLADE-ON-PETIOLE (BOP) genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate adaptors in a CUL3BOP1/BOP2 E3 ubiquitin ligase complex, targeting PIF4 proteins for ubiquitination and subsequent degradation.

Abstract 14112: Apabetalone (RVX-208) Reduces ACE2 Expression in Human Cell Culture Systems, Which Could Attenuate SARS-CoV-2 Viral Entry

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Dean Gilham ◽  
Li Fu ◽  
Laura M Tsujikawa ◽  
Brooke Rakai ◽  
Sylwia Wasiak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trials ◽  
Human Cell ◽  
Viral Entry ◽  
Host Cells ◽  
Hepatoma Cell Line ◽  
Bet Inhibitors ◽  
Bet Protein ◽  
The Impact ◽  
Ace2 Gene

Introduction: Apabetalone is an orally available small molecule that affects gene transcription by inhibiting BET protein interactions with acetylated histones and transcription factors. Apabetalone is in late stage clinical development for the treatment of cardiovascular disease (CVD). Clinical trials show apabetalone is well tolerated and has a favorable safety profile. Recent evidence indicates this drug could be repurposed to treat COVID-19 by reducing expression of the angiotensin converting enzyme 2 (ACE2) receptor that is essential for SARS-CoV-2 viral entry and/or disrupting viral protein E interaction with BET proteins. The spike protein of SARS-CoV-2 engages with human ACE2 expressed in multiple organs such as lung, liver, kidney, heart and intestine to initiate infection in host cells. Ultimately, the infection leads to life threatening complications in COVID-19 patients. Hypothesis: Apabetalone downregulates ACE2 expression to protect human cells from SARS-CoV-2. Methods: Primary human kidney tubular epithelial cells (RPTEC) were stimulated with TNFα and co-treated with apabetalone overnight. Primary human hepatocytes (PHH) and the hepatoma cell line, HepG2, were treated up to 96 h with apabetalone or other BET inhibitors (BETi). Gene expression was analyzed by microarray or RT-PCR. Human aortic endothelial cells (HAEC) were pretreated with apabetalone for 1 h followed by TNFα stimulation for another 1h. Chromatin occupancy of the BET protein BRD4 was examined by ChIP-seq. Results: In RPTEC, 5μM apabetalone downregulated ACE2 mRNA by 50%. Apabetalone dose dependently reduced ACE2 gene expression in HepG2 or PHH from 3 independent donors by up to 90%. JQ1 is a pan BET inhibitor, whereas MZ1 promotes BET protein degradation. Both JQ1 and MZ1 treatments downregulated ACE2 in liver cells, indicating an on target BETi effect. In HAEC, apabetalone abolished BRD4 occupancy at an enhancer in proximity to the ACE2 gene. Conclusions: Apabetalone reduces ACE2 gene expression in multiple human cell types. ACE2 expression may be regulated by adjacent BRD4 enhancer occupancy. The impact of apabetalone on SARS-CoV-2 life cycle is under investigation. The results will provide mechanistic support for potential COVID-19 clinical trials.

scholarly journals A functional motif of long noncoding RNA Nron against osteoporosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fujun Jin ◽  
Junhui Li ◽  
Yong-Biao Zhang ◽  
Xiangning Liu ◽  
Mingxiang Cai ◽  
...  
Keyword(s):  
Side Effects ◽  
Bone Resorption ◽  
Bone Loss ◽  
Transgenic Mice ◽  
Bone Mass ◽  
Ubiquitin Ligase ◽  
Noncoding Rna ◽  
E3 Ubiquitin Ligase ◽  
Noncoding Rnas ◽  
Functional Motif

AbstractLong noncoding RNAs are widely implicated in diverse disease processes. Nonetheless, their regulatory roles in bone resorption are undefined. Here, we identify lncRNA Nron as a critical suppressor of bone resorption. We demonstrate that osteoclastic Nron knockout mice exhibit an osteopenia phenotype with elevated bone resorption activity. Conversely, osteoclastic Nron transgenic mice exhibit lower bone resorption and higher bone mass. Furthermore, the pharmacological overexpression of Nron inhibits bone resorption, while caused apparent side effects in mice. To minimize the side effects, we further identify a functional motif of Nron. The delivery of Nron functional motif to osteoclasts effectively reverses bone loss without obvious side effects. Mechanistically, the functional motif of Nron interacts with E3 ubiquitin ligase CUL4B to regulate ERα stability. These results indicate that Nron is a key bone resorption suppressor, and the lncRNA functional motif could potentially be utilized to treat diseases with less risk of side effects.

scholarly journals Nonhost Resistance of Tomato to the Bean Pathogen Pseudomonas syringae pv. syringae B728a Is Due to a Defective E3 Ubiquitin Ligase Domain in AvrPtoBB728a

2013 ◽  
Vol 26 (4) ◽  
pp. 387-397 ◽  
Author(s):  
Ching-Fang Chien ◽  
Johannes Mathieu ◽  
Chun-Hua Hsu ◽  
Patrick Boyle ◽  
Gregory B. Martin ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Amino Acid Identity ◽  
Nonhost Resistance ◽  
Plant Host ◽  
Terminal Domain ◽  
Tomato Cultivars

The bean pathogen Pseudomonas syringae pv. syringae B728a expresses homologs of the type III effectors AvrPto and AvrPtoB, either of which can trigger resistance in tomato cultivars expressing Pto and Prf genes. We found that strain B728a also elicits nonhost resistance in tomato cultivars VFNT Cherry and Moneymaker that lack Pto but express other members of the Pto family (e.g., SlFen and SlPtoC). Here, we show that the AvrPtoB homolog from B728a, termed AvrPtoBB728a (also known as HopAB1), is recognized by ‘VFNT Cherry’ and ‘Moneymaker’ when the effector is expressed in P. syringae pv. syringae 61, a strain lacking the avrPto or avrPtoB homolog. Using a gene-silencing approach, this recognition was shown to involve one or more Pto family members and Prf. AvrPtoBB728a interacted with SlFen, SlPtoC, and SlPtoD, in addition to Pto, in a yeast two-hybrid assay. In P. syringae pv. tomato DC3000, the C-terminal domain of AvrPtoB is an E3 ubiquitin ligase that ubiquitinates Fen, causing its degradation and leading to disease susceptibility. Although the C-terminal domain of AvrPtoBB728a shares 69% amino acid identity with that of AvrPtoB, we found that it has greatly reduced E3 ligase activity and is unable to ubiquitinate Fen in an in vitro ubiquitination assay. Thus, the nonhost resistance of ‘VFNT Cherry’ and ‘Moneymaker’ to B728a appears to be due to recognition of AvrPtoBB728 as a result of the effector's reduced E3 ligase activity, which prevents it from facilitating degradation of a Pto family member. We speculate that the primary plant host of B728a lacks a Fen-like protein and that, therefore, the E3 ligase of AvrPtoBB728 was unnecessary for pathogenicity and has diverged and become ineffective.

scholarly journals The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins

2019 ◽  
Vol 294 (28) ◽  
pp. 10900-10912 ◽  
Author(s):  
Wonjin Yoo ◽  
Eun-Bee Cho ◽  
Sungjoo Kim ◽  
Jong-Bok Yoon
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
E3 Ubiquitin Ligase ◽  
Secretory Proteins ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Lysine Residues ◽  
Subsequent Degradation ◽  
And Function

The E3 ubiquitin ligase membrane-associated ring-CH–type finger 2 (MARCH2) is known to be involved in intracellular vesicular trafficking, but its role in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi compartments is largely unknown. Human ER–Golgi intermediate compartment protein 2 (ERGIC2) and ERGIC3 are orthologs of Erv41 and Erv46 in yeast, proteins that form a heteromeric complex, cycle between the ER and Golgi, and function as cargo receptors in both anterograde and retrograde protein trafficking. Here, we report that MARCH2 directs ubiquitination and subsequent degradation of ERGIC3 and that MARCH2 depletion increases endogenous ERGIC3 levels. We provide evidence that the lysine residues at positions 6 and 8 of ERGIC3 are the major sites of MARCH2-mediated ubiquitination. Of note, MARCH2 did not significantly decrease the levels of an ERGIC3 variant with lysine-to-arginine substitutions at residues 6 and 8. We also show that ERGIC3 binds to itself or to ERGIC2, whereas ERGIC2 is unable to interact with itself. Our results indicate that α1-antitrypsin and haptoglobin are likely to be cargo proteins of ERGIC3. We further observed that α1-antitrypsin and haptoglobin specifically bind to ERGIC3 and that ERGIC3 depletion decreases their secretion. Moreover, MARCH2 reduced secretion of α1-antitrypsin and haptoglobin, and coexpression of the ubiquitination-resistant ERGIC3 variant largely restored their secretion, suggesting that MARCH2-mediated ERGIC3 ubiquitination is the major cause of the decrease in trafficking of ERGIC3-binding secretory proteins. Our findings provide detailed insights into the regulation of the early secretory pathway by MARCH2 and into ERGIC3 function.

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