35 research outputs found
PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network
Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development
Human De-Etiolated-1 Regulates c-Jun by Assembling a CUL4A Ubiquitin Ligase
Arabidopsis thaliana De-etiolated-1 (AtDET1) is a highly conserved protein, with orthologs in vertebrate and invertebrate organisms. AtDET1 negatively regulates photomorphogenesis, but its biochemical mechanism and function in other species are unknown. We report that human DET1 (hDET1) promotes ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun by assembling a multisubunit ubiquitin ligase containing DNA Damage Binding Protein-1 (DDB1), cullin 4A (CUL4A), Regulator of Cullins-1 (ROC1), and constitutively photomorphogenic-1. Ablation of any subunit by RNA interference stabilized c-Jun and increased c-Jun–activated transcription. These findings characterize a c-Jun ubiquitin ligase and define a specific function for hDET1 in mammalian cells
Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease
Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-B regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet\u27s disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-B signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IBα and nuclear translocation of the NF-B p65 subunit together with increased expression of NF-B-mediated proinflammatory cytokines. A20 restricts NF-B signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-B-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease
Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation
Targeted
protein degradation with proteolysis targeting chimeras
(PROTACs) is a powerful therapeutic modality for eliminating disease-causing
proteins through targeted ubiquitination and proteasome-mediated degradation.
Most PROTACs have exploited substrate receptors of Cullin-RING E3
ubiquitin ligases such as cereblon and VHL. Whether core, shared,
and essential components of the Cullin-RING E3 ubiquitin ligase complex
can be used for PROTAC applications remains less explored. Here, we
discovered a cysteine-reactive covalent recruiter EN884 against the
SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex.
We further showed that this recruiter can be used in PROTAC applications
to degrade neo-substrate proteins such as BRD4 and the androgen receptor
in a SKP1- and proteasome-dependent manner. Our studies demonstrate
that core and essential adapter proteins within the Cullin-RING E3
ubiquitin ligase complex can be exploited for targeted protein degradation
applications and that covalent chemoproteomic strategies can enable
recruiter discovery against these targets
Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation
Targeted
protein degradation with proteolysis targeting chimeras
(PROTACs) is a powerful therapeutic modality for eliminating disease-causing
proteins through targeted ubiquitination and proteasome-mediated degradation.
Most PROTACs have exploited substrate receptors of Cullin-RING E3
ubiquitin ligases such as cereblon and VHL. Whether core, shared,
and essential components of the Cullin-RING E3 ubiquitin ligase complex
can be used for PROTAC applications remains less explored. Here, we
discovered a cysteine-reactive covalent recruiter EN884 against the
SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex.
We further showed that this recruiter can be used in PROTAC applications
to degrade neo-substrate proteins such as BRD4 and the androgen receptor
in a SKP1- and proteasome-dependent manner. Our studies demonstrate
that core and essential adapter proteins within the Cullin-RING E3
ubiquitin ligase complex can be exploited for targeted protein degradation
applications and that covalent chemoproteomic strategies can enable
recruiter discovery against these targets
Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation
Targeted
protein degradation with proteolysis targeting chimeras
(PROTACs) is a powerful therapeutic modality for eliminating disease-causing
proteins through targeted ubiquitination and proteasome-mediated degradation.
Most PROTACs have exploited substrate receptors of Cullin-RING E3
ubiquitin ligases such as cereblon and VHL. Whether core, shared,
and essential components of the Cullin-RING E3 ubiquitin ligase complex
can be used for PROTAC applications remains less explored. Here, we
discovered a cysteine-reactive covalent recruiter EN884 against the
SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex.
We further showed that this recruiter can be used in PROTAC applications
to degrade neo-substrate proteins such as BRD4 and the androgen receptor
in a SKP1- and proteasome-dependent manner. Our studies demonstrate
that core and essential adapter proteins within the Cullin-RING E3
ubiquitin ligase complex can be exploited for targeted protein degradation
applications and that covalent chemoproteomic strategies can enable
recruiter discovery against these targets