Biology subjects

Rape, M.

Publications and source records attributed to Rape, M..

12 recordsLinked to original sources

bioRxiv2026

Systemic degradation of repressive transcription factors gates gene expression and cell fate specification

While proteasomes are best known for eliminating defective proteins or turning off signaling pathways, they also enable crucial cellular activities. Critical among these, proteasomes allow cells to initiate gene expression, but underlying targets and regulatory mechanisms remain poorly understood. Here, we report that proteasomes drive the systemic degradation of repressive trans-cription factors to eject TLE/Groucho-family co-repressors from chromatin and thereby constantly free transcription start sites for activator binding. This circuitry requires the E3 ligase SCFFBXL14, which modifies its targets dependent on presentation by TLEs, but independently of their identity. The continuous cycling of co-repressors off chromatin, as achieved by systemic turnover of a protein family, is essential for stem cells to translate developmental cues into lineage-specific gene expression, and it is disrupted by cancer mutations in TLE1 that impair SCFFBXL14-recruit-ment. We conclude that systemic degradation of repressive transcription factors establishes co-repressor dynamics required for genes expression and cell fate specification.

biochemistry↗

bioRxiv2026

Localized heme sensing through a ternary molecular glue

Molecular glues are an emerging class of therapeutics that stabilize binary interactions and there-by rewire disease-relevant protein networks. Whether glues can integrate additional information to orchestrate signaling beyond initial complex formation is unknown. Here, we report that cells use an endogenous glue strategy to sense heme, an essential metabolite with deleterious pro-oxidant properties. Distinct from other glues, heme bridges three polypeptides to trigger degradation of the transcriptional repressor BACH1 through cytoplasmic, but not mitochondrial, CUL2FEM1B. This mechanism allows cells to eliminate toxic heme in the cytoplasm by inducing expression of the heme-degrading oxygenase HMOX1, yet ignore mitochondrial heme destined for function in the electron transport chain. While protective in healthy cells, ternary glue signaling creates a therapeutic vulnerability for Acute Myeloid Leukemias dependent on high rates of ETC assembly. Molecular glues can therefore drive assembly of higher-order complexes to establish localized signaling, which offers unexplored opportunities for induced proximity therapeutics.

biochemistry↗

bioRxiv2026

Metabolic glues as a means of purine sensing and chemotherapeutic response

Molecular glues stabilize weak interactions to impart novel functionalities onto complexes. While plant hormones or drugs are known molecular glues, it is still unknown whether this modality provides endogenous regulation in human cells. Here, we show that purine nucleotides are molecular glues that tether the rate-limiting enzyme of purine biosynthesis, phosphoribosyl-pyrophosphate-amidotransferase (PPAT), to its inhibitor NUDT5. This mechanism allows cells to sense purine levels and establish essential feedback control of their synthesis. Thiopurine chemotherapeutics, in clinical use since the 1950s, act as molecular glues of the same complex, but adopt unique orientations for enhanced function. Distinct from the recognition of many therapeutic glues, metabolic glue pockets can adjust their conformation to significant compound alterations and thereby enable increasing glue potency without sacrificing specificity. Our findings therefore identify endogenous metabolic glues as a mode of nutrient sensing that can be exploited to obtain compounds that rewire metabolic pathways for therapeutic benefit.

biochemistry↗

bioRxiv2026

Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

Medulloblastoma, the most common malignant pediatric brain tumor, arises from developmental aberrations of cerebellar precursor cells. The CUL3-RING ubiquitin ligase adaptor KBTBD4 is recurrently mutated in medulloblastoma subgroups 3 and 4. While KBTBD4 mutations confer a gain-of-function phenotype leading to aberrant degradation of transcriptional repressors, endogenous targets of this E3 ligase remain unknown. Here, we identify the PP2A-A scaffolding subunit of the PP2A phosphatase as a CRL3KBTBD4 substrate. Using a combination of proteomics, cell biology, biochemical reconstitution, and cryo-EM structural analyses, we show that CRL3KBTBD4 mediates orphan quality control by targeting free PP2A-A for degradation to safeguard phosphatase activity. Loss of KBTBD4 or its mutation in medulloblastoma cause PP2A-A accumulation, hence affecting phospho-dependent signaling pathways in cancer development. Disease mutations in KBTBD4 thus elicit a dual phenotype: gain-of-function degradation of transcriptional repressors combined with loss of PP2A quality control, which dysregulates multiple signaling events implicated in cancer, including telomere length regulation.

cell biology↗

bioRxiv2025

Translation-driven temporal control for intertwined protein assembly

Protein complexes are essential to cells. However, how structurally intertwined protein subunits can assemble faithfully is poorly understood. Here, we reveal a "temporal control" mechanism driven by coupled ribosomes to form intertwined dimers. Using Disome Selective Profiling and optical tweezers, we show that the BTB domains of KEAP1, KLHL12, and PATZ1 form stable closed states as monomers, thus impeding proposed domain-swapping assembly routes. By contrast, the timed emergence of nascent chain segments during translation enables alternative folding-assembly pathways that bypass the closed monomeric state. Analysis indicates that this mechanism works in concert with dimerization quality control by the E3 ligase SCF-FBXL17, and is relevant across the BTB domain family. This study shows that ribosome cooperation expands the range of possible protein architectures.

molecular biology↗

bioRxiv2025

Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

Metazoan development requires that cells adopt specific identities at the right time and place with-in an embryo. Central to the success of this process are posttranslational modifications that control the activity, stability or localization of crucial transducers of differentiation signals. During muscle development, modification of proteins with ubiquitin is known to play an important role, but the enzymatic machinery of ubiquitylation that drives myogenesis remains incompletely understood. Here, we identify CUL3BTBD9 as an E3 ubiquitin ligase that is essential for myogenesis in vitro. CUL3BTBD9 binds and ubiquitylates CAV1, the central component of caveolae that modulate insulin signaling during muscle formation. CUL3BTBD9 and CAV1 are required for insulin-dependent activation of the AKT kinase in myoblasts, thereby safeguarding the ability of muscle precursors to respond to insulin signals. Together, this work identifies CUL3BTBD9 as a regulator of myogenesis that acts by modulating plasma-membrane localized events critical for cell fate specification.

cell biology↗

bioRxiv2025

Poxvirus attack of anti-viral defense pathways unleashes an effector-triggered NF-κB response

Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or "effectors". To discover novel ETI pathways in mammals, we developed a screening approach in which individual virulence factors are expressed in human monocytes and transcriptional responses are assessed by RNA-seq. Using this approach, we identify a poxvirus effector, myxoma virus M3.1, which elicits an anti-viral NF-{kappa}B response. We find that NF-{kappa}B is unleashed by an ETI pathway that senses M3.1 attack of two anti-viral complexes: ZAP and TBK1. NF-{kappa}B activation occurs because the proteins inhibited by M3.1-- N4BP1, ZC3H12A, and TBK1--are negative regulators of NF-{kappa}B. Our results illustrate how negative regulators can function as pathogen sensors and establish a systematic approach for the discovery of ETI pathways.

immunology↗

bioRxiv2024

The molecular basis of integrated stress response silencing

Chronic stress response activation impairs cell survival and causes devastating degenerative diseases. To counteract this, cells deploy dedicated silencing factors, such as the E3 ligase SIFI that terminates the mitochondrial stress response. How a single enzyme can sense stress across cells and elicit timely stress response inactivation is poorly understood. Here, we report the structure of human SIFI, which revealed how this 1.3MDa complex can target hundreds of proteins for accurate stress response silencing. SIFI attaches the first ubiquitin to substrates using flexible domains within an easily accessible scaffold, yet builds linkage-specific ubiquitin chains at distinct, sterically restricted elongation modules in its periphery. Ubiquitin handover via a ubiquitin-like domain couples versatile substrate modification to precise chain elongation. Stress response silencing therefore exploits a catalytic mechanism that is geared to process many diverse proteins and hence allows a single enzyme to monitor and, if appropriate, modulate a complex cellular state.

biochemistry↗

bioRxiv2024

Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12

The oxidative stress response is centered on the transcription factor NRF2 and protects cells from reactive oxygen species (ROS). While ROS inhibit the E3 ligase CUL3KEAP1 to stabilize NRF2 and elicit antioxidant gene expression, cells recovering from stress must rapidly reactivate CUL3KEAP1 to prevent reductive stress and oxeiptosis-dependent cell death. How cells restore efficient NRF2-degradation upon ROS clearance remains poorly understood. Here, we identify TRIP12, an E3 ligase dysregulated in Clark-Baraitser Syndrome and Parkinsons Disease, as a component of the oxidative stress response. TRIP12 is a ubiquitin chain elongation factor that cooperates with CUL3KEAP1 to ensure robust NRF2 degradation. In this manner, TRIP12 accelerates stress response silencing as ROS are being cleared, but limits NRF2 activation during stress. The need for dynamic control of NRF2-degradation therefore comes at the cost of diminished stress signaling, suggesting that TRIP12 inhibition could be used to treat degenerative pathologies characterized by ROS accumulation.

biochemistry↗

bioRxiv2022

Orphan quality control shapes network dynamics and gene expression

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb crucial interactions between network components often result in disease, but how the composition and dynamics of complex networks are established is unknown. Here, we identify the tumor suppressor E3 ligase UBR5 as a quality control enzyme that helps degrade unpaired subunits of multiple transcription factors that operate within a single network. By constantly turning over orphan subunits, UBR5 forces cells to continuously replenish network components through new protein synthesis. The resulting cycles of transcription factor synthesis and degradation allow cells to effectively execute the gene expression program, while remaining susceptible to environmental signals. We conclude that orphan quality control plays an essential role in establishing the dynamics of protein networks, which may explain the conserved need for protein degradation in transcription and offers unique opportunities to modulate gene expression in disease.

biochemistry↗

bioRxiv2021

Large cells activate global protein degradation to maintain cell size homeostasis

Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. This tight control of cell size involves both cell size checkpoints, which delay cell cycle progression in small cells, and size-dependent regulation of mass accumulation rates. While we previously identified the p38 MAPK pathway as a key regulator of the mammalian cell size checkpoint, the mechanism of size-dependent growth rate regulation has remained elusive. Here, we quantified global rates of protein synthesis and degradation in cells of varying sizes, both under unperturbed conditions and in response to perturbations that trigger size-dependent compensatory growth slowdown. We found that protein synthesis rates scale proportionally with cell size across cell cycle stages and experimental conditions. In contrast, oversized cells that undergo compensatory growth slowdown exhibit a superlinear increase in proteasome-mediated protein degradation, with accelerated protein turnover per unit mass, suggesting activation of the proteasomal degradation pathway. Both nascent and long-lived proteins contribute to the elevated protein degradation during compensatory growth slowdown, with long-lived proteins playing a crucial role at the G1/S transition. Notably, large G1/S cells exhibit particularly high efficiency in protein degradation, surpassing that of similarly sized or larger cells in S and G2, coinciding with the timing of the most stringent size control in animal cells. These results collectively suggest that oversized cells reduce their growth efficiency by activating global proteasome-mediated protein degradation to promote cell size homeostasis.

cell biology↗

bioRxiv2021

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting Cys186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to specific FEM1B- and proteasome-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications.

biochemistry↗