A Novel Degron-mediated Degradation of the RTG Pathway Regulator, Mks1p, by SCFGrr1

Yeast cells respond to mitochondrial dysfunction by altering the expression of a subset of nuclear genes, a process known as retrograde signaling (RS). RS terminates with two transcription factors, Rtg1p and Rtg3p. One positive regulator, Rtg2p, and four negative regulators, Lst8p, Mks1p, and the redundant 14-3-3 proteins, Bmh1p and Bmh2p, control RS upstream of Rtg1/3p. Mks1p is negatively regulated by binding to Rtg2p and positively regulated when bound to Bmh1/2p. Here we report that Grr1p, a component of the SCFGrr1 E3 ubiquitin ligase, modulates RS by affecting Mks1p levels. Grr1p polyubiquitinates Mks1p not bound to either Rtg2p or to Bmh1/2p, targeting it for degradation. An acidic domain region of Mks1p constitutes the portable Mks1p degron sequence. We have isolated dominant mutations in Grr1p leading to increased Mks1p degradation. These mutations result in a gain of positive charge on the concave surface of the leucine rich repeat (LRR) domain of Grr1p, the proposed substrate binding site. We propose that Mks1p is a central player of RS and is acted upon by multiple regulators of the pathway.

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Sequential ubiquitination of NLRP3 by RNF125 and Cbl-b limits inflammasome activation and endotoxemia

Journal of Experimental Medicine ◽

10.1084/jem.20182091 ◽

2020 ◽

Vol 217 (4) ◽

Cited By ~ 7

Author(s):

Juan Tang ◽

Sha Tu ◽

Guoxin Lin ◽

Hui Guo ◽

Chengkai Yan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Lethal Dose ◽

E3 Ubiquitin Ligase ◽

Inflammasome Activation ◽

Dependent Manner ◽

Leucine Rich Repeat ◽

Nlrp3 Inflammasome Activation ◽

Repeat Domain ◽

Nlrp3 Inflammasomes ◽

Lrr Domain

Aberrant NLRP3 inflammasome activation contributes to the development of endotoxemia. The importance of negative regulation of NLRP3 inflammasomes remains poorly understood. Here, we show that the E3 ubiquitin ligase Cbl-b is essential for preventing endotoxemia induced by a sub-lethal dose of LPS via a caspase-11/NLRP3–dependent manner. Further studies show that NLRP3 undergoes both K63- and K48-linked polyubiquitination. Cbl-b binds to the K63-ubiquitin chains attached to the NLRP3 leucine-rich repeat domain (LRR) via its ubiquitin-associated region (UBA) and then targets NLRP3 at K496 for K48-linked ubiquitination and proteasome-mediated degradation. We also identify RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked ubiquitination of the NLRP3 LRR domain. Therefore, NLRP3 is sequentially ubiquitinated by K63- and K48-linked ubiquitination, thus keeping the NLRP3 inflammasomes in check and restraining endotoxemia.

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Treatment of Lymphoid and Myeloid Malignancies by Immunomodulatory Drugs

Cardiovascular & Haematological Disorders - Drug Targets ◽

10.2174/1871529x18666180522073855 ◽

2019 ◽

Vol 19 (1) ◽

pp. 51-78 ◽

Cited By ~ 3

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.

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OP0119 The CRL4 Cereblon E3 Ubiquitin Ligase Modulator CC-220 Induces Degradation of the Transcription Factors Aiolos and Ikaros: Immunomodulation in Healthy Volunteers and Relevance to Systemic Lupus Erythematosus

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2015-eular.3498 ◽

2015 ◽

Vol 74 (Suppl 2) ◽

pp. 113.2-113 ◽

Cited By ~ 2

Author(s):

P. Schafer ◽

Y. Ye ◽

L. Wu ◽

J. Kosek ◽

Z. Yang ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Transcription Factors ◽

Healthy Volunteers ◽

Ubiquitin Ligase ◽

Lupus Erythematosus ◽

E3 Ubiquitin Ligase ◽

Systemic Lupus

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SUSA2 is an F-box protein required for autoimmunity mediated by paired NLRs SOC3-CHS1 and SOC3-TN2

Nature Communications ◽

10.1038/s41467-020-19033-z ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Wanwan Liang ◽

Meixuezi Tong ◽

Xin Li

Keyword(s):

Ubiquitin Ligase ◽

Higher Plants ◽

Interaction Analysis ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Pairwise Interaction ◽

Leucine Rich Repeat ◽

Scf Complex ◽

F Box Protein ◽

Effector Recognition

Abstract Both higher plants and mammals rely on nucleotide-binding leucine-rich repeat (NLR) immune receptors to detect pathogens and initiate immunity. Upon effector recognition, plant NLRs oligomerize for defense activation, the mechanism of which is poorly understood. We previously showed that disruption of the E3 ligase, Senescence-Associated E3 Ubiquitin Ligase 1 (SAUL1) leads to the activation of the NLR SOC3. Here, we report the identification of suppressor of saul1 2 (susa2) and susa3 from the saul1-1 suppressor screen. Pairwise interaction analysis suggests that both SUSA proteins interact with components of an SCFSUSA2 E3 ligase complex as well as CHS1 or TN2, truncated NLRs that pair with SOC3. susa2-2 only suppresses the autoimmunity mediated by either CHS1 or TN2, suggesting its specific involvement in SOC3-mediated immunity. In summary, our study indicates links between plant NLRs and an SCF complex that may enable ubiquitination and degradation of unknown downstream components to activate defense.

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The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

mSphere ◽

10.1128/msphere.00014-15 ◽

2015 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Frédéric Dallaire ◽

Sabrina Schreiner ◽

G. Eric Blair ◽

Thomas Dobner ◽

Philip E. Branton ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Dna Synthesis ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Viral Gene Expression ◽

Adenovirus Vector ◽

Human Adenovirus ◽

Viral Gene ◽

E2f Transcription Factors

ABSTRACT During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication.

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Functions of COP1/SPA E3 Ubiquitin Ligase Mediated by MpCRY in the Liverwort Marchantia Polymorpha under Blue Light

International Journal of Molecular Sciences ◽

10.3390/ijms23010158 ◽

2021 ◽

Vol 23 (1) ◽

pp. 158

Author(s):

Li Zhang ◽

Tianhong Li ◽

Shengzhong Su ◽

Hao Peng ◽

Sudi Li ◽

...

Keyword(s):

Transcription Factors ◽

Blue Light ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Land Plants ◽

Marchantia Polymorpha ◽

Regulatory Pathway ◽

Knock Out

COP1/SPA1 complex in Arabidopsis inhibits photomorphogenesis through the ubiquitination of multiple photo-responsive transcription factors in darkness, but such inhibiting function of COP1/SPA1 complex would be suppressed by cryptochromes in blue light. Extensive studies have been conducted on these mechanisms in Arabidopsis whereas little attention has been focused on whether another branch of land plants bryophyte utilizes this blue-light regulatory pathway. To study this problem, we conducted a study in the liverwort Marchantia polymorpha and obtained a MpSPA knock-out mutant, in which Mpspa exhibits the phenotype of an increased percentage of individuals with asymmetrical thallus growth, similar to MpCRY knock-out mutant. We also verified interactions of MpSPA with MpCRY (in a blue light-independent way) and with MpCOP1. Concomitantly, both MpSPA and MpCOP1 could interact with MpHY5, and MpSPA can promote MpCOP1 to ubiquitinate MpHY5 but MpCRY does not regulate the ubiquitination of MpHY5 by MpCOP1/MpSPA complex. These data suggest that COP1/SPA ubiquitinating HY5 is conserved in Marchantia polymorpha, but dissimilar to CRY in Arabidopsis, MpCRY is not an inhibitor of this process under blue light.

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Parkin: a multifaceted ubiquitin ligase

Biochemical Society Transactions ◽

10.1042/bst0340749 ◽

2006 ◽

Vol 34 (5) ◽

pp. 749-753 ◽

Cited By ~ 82

Author(s):

D.J. Moore

Keyword(s):

Ubiquitin Ligase ◽

Dopaminergic Neurons ◽

Autosomal Recessive ◽

E3 Ubiquitin Ligase ◽

Gene Products ◽

Biochemical Pathway ◽

Leucine Rich Repeat ◽

Parkin Gene ◽

Protein Substrates ◽

Phosphatase And Tensin Homologue

Mutations in the parkin gene are a common cause of autosomal recessive early-onset parkinsonism. Parkin functions as an E3 ubiquitin ligase where it can polyubiquitinate a number of its protein substrates, thus targeting them for degradation by the 26 S proteasomal complex. Recent studies have demonstrated that alternative modes of parkin-mediated ubiquitination may serve other non-degradative regulatory roles. In addition, parkin appears to function as a multipurpose neuroprotectant in a number of toxic paradigms. Coupled with these observations, parkin may integrate other gene products associated with parkinsonism, including α-synuclein, LRRK2 (leucine-rich repeat kinase 2), DJ-1 and PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], into a common biochemical pathway of potential relevance to disease pathogenesis. Parkin therefore represents a unique multifaceted ubiquitin ligase consistent with an important housekeeping role in maintaining the integrity or survival of dopaminergic neurons.

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Targeting oncoproteins with a positive selection assay for protein degraders

Science Advances ◽

10.1126/sciadv.abd6263 ◽

2021 ◽

Vol 7 (6) ◽

pp. eabd6263

Author(s):

Vidyasagar Koduri ◽

Leslie Duplaquet ◽

Benjamin L. Lampson ◽

Adam C. Wang ◽

Amin H. Sabet ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Transcription Factors ◽

Small Molecules ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Signal To Noise ◽

Independent Manner ◽

Intracellular Proteins ◽

Oncogenic Transcription Factor

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9–based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

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E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603310113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7557-7562 ◽

Cited By ~ 11

Author(s):

Yan Zhang ◽

Shigetoshi Yokoyama ◽

John C. Herriges ◽

Zhen Zhang ◽

Randee E. Young ◽

...

Keyword(s):

Transcription Factors ◽

Ubiquitin Ligase ◽

Protein Level ◽

E3 Ubiquitin Ligase ◽

Transcript Level ◽

Branching Morphogenesis ◽

Normal Lung ◽

Lung Epithelium ◽

E3 Ligases ◽

Lung Branching

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.

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