Biology subjects

Crews, C. M.

Publications and source records attributed to Crews, C. M..

5 recordsLinked to original sources

bioRxiv2024

An atypical E3 ligase safeguards the ribosome during nutrient stress

Metabolic stress must be effectively mitigated for the survival of cells and organisms. Ribosomes have emerged as signaling hubs that sense metabolic perturbations and coordinate responses that either restore homeostasis or trigger cell death. As yet, the mechanisms governing these cell fate decisions are not well understood. Here, we report an unexpected role for the atypical E3 ligase HOIL-1 in safeguarding the ribosome. We find HOIL-1 mutations associated with cardiomyopathy broadly sensitize cells to nutrient and translational stress. These signals converge on the ribotoxic stress sentinel ZAK. Mechanistically, mutant HOIL-1 excludes a ribosome quality control E3 ligase from its functional complex and remodels the ribosome ubiquitin landscape. This quality control failure renders glucose starvation ribotoxic, precipitating a ZAK-ATF4-xCT-driven noncanonical cell death. We further show HOIL-1 loss exacerbates cardiac dysfunction under pressure overload. These data reveal an unrecognized ribosome signaling axis and a molecular circuit controlling cell fate during nutrient stress.

cell biology↗

bioRxiv2023

Regulated Induced Proximity Targeting Chimeras (RIPTACs): a Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively

While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities. Here, we describe a proof-of-concept study using novel heterobifunctional small molecules called Regulated Induced Proximity Targeting Chimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein. Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

cancer biology↗

bioRxiv2022

Hijacking Methyl Reader proteins for Nuclear-Specific Protein Degradation

Targeted protein degradation (TPD) by PROTACs is a promising strategy to control disease-causing protein levels within the cell. While TPD is emerging as an innovative drug discovery paradigm, there are currently only a limited number of E3 ligase: ligand pairs that are employed to induce protein degradation. Herein, we report a novel approach to induce protein degradation by hijacking a methyl reader: E3 ligase complex. L3MBTL3 is a methyl lysine reader protein that binds to the Cul4DCAF5 E3 ligase complex and targets methylated proteins for proteasomal degradation. By co-opting this natural mechanism, we report the design and biological evaluation of L3MBTL3-recruiting PROTACs and demonstrate nuclear-specific degradation of FKBP12 and BRD2. We envision this as a generalizable approach to utilize other reader protein-associated E3 ligase complexes in PROTAC design to expand the E3 ligase toolbox and explore the full potential of TPD.

pharmacology and toxicology↗

bioRxiv2020

Targeted Degradation of Transcription Factors by TRAFTACs: Transcription Factor Targeting Chimeras

Many diseases, including cancer, stem from aberrant activation and overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity are able to be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription-factor degradation. Herein, we show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription-factor of interest (TOI) and to HaloTag fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-{kappa}B and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins with minor changes to the chimeric oligonucleotide.

bioengineering↗

bioRxiv2020

Mutant-selective Degradation by BRAF-targeting PROTACs

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we successfully achieve sub-nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable using degradation-based therapies by targeting mutant BRAF-driven cancers while sparing BRAFWT and thus expanding the therapeutic window using a new anti-tumor drug modality.

cancer biology↗