Biology subjects

Emanuele, M. J.

Publications and source records attributed to Emanuele, M. J..

8 recordsLinked to original sources

bioRxiv2025

Charting the Multi-level Molecular Response to Palbociclib in ER-Positive Breast Cancer

The addition of CDK4/6 inhibitors to endocrine therapy has significantly improved outcomes in HR+/HER2-breast cancer. However, variable patient responses and acquired resistance remain a clinical challenge. We therefore defined the comprehensive molecular response to palbociclib, the most clinically used CDK4/6 inhibitor. Global analyses of gene expression, protein abundance, splicing, and chromatin accessibility revealed broad patterns and specific changes that result from CDK4/6-inhibition in breast cancer cells. We uncovered unexpected feedback between CDK4/6 and estrogen-response signaling, which has clear clinical implications. We also revealed a widespread alternative splicing program that partially overlapped with genes whose expression is regulated, and which is expected to impact protein function. These molecular changes nominated combination therapies that interfere with the activation of CDKs or ER. Accordingly, co-targeting CDK7, which regulates CDK2, CDK4/6 and ER, additively impacted cell fitness. Collectively, these data reveal a complex, multi-tiered response to CDK4/6 inhibition, with implications for therapeutic efficacy.

molecular biology↗

bioRxiv2025

Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22

Targeted protein degradation (TPD) through the ubiquitin-proteasome system is driven by compound-mediated polyubiquitination of a protein-of-interest by an E3 ubiquitin (Ub) ligase. To date, relatively few E3s have been successfully utilized for TPD and the governing principles of functional ternary complex formation between the E3, degrader, and protein target remain elusive. FBXO22 has recently been harnessed by several groups to target different proteins for degradation. FBXO22 recruitment has been enabled through degraders that covalently modify its cysteine residues. Here, we reveal that the aldehyde derivative of UNC10088 promotes cooperative binding of FBXO22 to NSD2, a histone methyltransferase and oncogenic protein, leading to a cryo-EM structure of the full SKP1-CUL1-F-box (SCF)-FBXO22 complex with NSD2. This structure revealed a conformational change in the FBXO22 loop surrounding C326, further exposing the cysteine for covalent recruitment. Additional medicinal chemistry efforts led to the discovery of benzaldehyde-based non-prodrug degraders that similarly engage C326 of FBXO22 and potently degrade NSD2. Furthermore, unlike many degraders, our molecules recruit NSD2 to a different surface of FBXO22 than the known FBXO22 substrate BACH1, allowing for concurrent complex formation and degradation of both the neosubstrate and endogenous substrates. Overall, we demonstrate the biochemical and structural basis for NSD2 degradation, revealing key principles for efficient and selective TPD by SCFFBXO22.

biochemistry↗

bioRxiv2025

Cdk1 and PP2A constitute a molecular switch controlling orderly degradation of atypical E2Fs

Dynamic oscillations in the phosphorylation and ubiquitination of key proliferative regulators are defining features of the eukaryotic cell cycle. Resetting the cell cycle at the mitosis-to-G1 transition requires activation of the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which ensures cell cycle irreversibility by targeting dozens of substrates for degradation, safeguarding genome integrity. However, the overall coupling of substrate phosphorylation with target recognition and degradation by the APC/C remains relatively unexplored. As a paradigm for further defining these rules, we focused on E2F7 and E2F8 - atypical E2F-family proteins that coordinate cell cycle gene expression by restraining the pro-proliferative transcriptional activity of E2F1. Leveraging complementary cell and cell-free systems, we demonstrate that flexible domains in the amino-termini of E2F7 and E2F8 contain APC/C recognition motifs adjacent to critical Thr residues, whose phosphorylation by Cdk1 is rate limiting for degradation. The removal of this phosphorylation by PP2A phosphatase serves as a molecular switch, coupling the degradation of E2F7 and E2F8 to the G1 phase, coinciding with the rise of E2F1. Collectively, these findings highlight a critical role for Cdk1-PP2A signaling in controlling the orderly degradation of APC/C substrates, ensuring precisely timed assembly of the transcriptional infrastructure that coordinates cell cycle commitment and progression.

cell biology↗

bioRxiv2025

Structural mechanism for recognition of E2F1 by the ubiquitin ligase adaptor Cyclin F

Cyclin F, a non-canonical member of the cyclin protein family, plays a critical role in regulating the precise transitions of cell-cycle events. Unlike canonical cyclins, which bind and activate cyclin-dependent kinases (CDKs), Cyclin F functions as a substrate receptor protein within the Skp1-Cullin-F box (SCF) E3 ubiquitin ligase complex, enabling the ubiquitylation of target proteins. The structural features that distinguish Cyclin F as a ligase adaptor and the mechanisms underlying its selective substrate recruitment over Cyclin A, which functions in complex with CDK2 at a similar time in the cell cycle, remain largely unexplored. We utilized single-particle cryo-electron microscopy to elucidate the structure of a Cyclin F-Skp1 complex bound to an E2F1 peptide. The structure and biochemical analysis reveal important differences in the substrate-binding site of Cyclin F compared to Cyclin A. Our findings expand on the canonical cyclin-binding motif (Cy or RxL) and highlight the importance of electrostatics at the E2F1 binding interface, which varies for Cyclin F and Cyclin A. Our results advance our understanding of E2F1 regulation and may inform the development of inhibitors targeting Cyclin F.

biochemistry↗

bioRxiv2023

Adaptive IRE1 Signaling Elicits T Cell Metabolic Remodeling and Tumor Control

The efficacy of cancer immunotherapies is limited by the metabolic instability of the tumor microenvironment (TME) that disables T cell antitumor immunity. Metabolic imbalances within the TME are sensed and responded to by stress sensors of the endoplasmic reticulum (ER) unfolded protein response (UPR). The UPR comprises three integrated signaling pathways harboring both adaptive and deleterious phases based on the extent and duration of cell stress. Here, we elucidate the differential contributions of adaptive and deleterious signaling downstream of the UPR IRE1 pathway in T cell-regulated tumor control. T cells in murine and patient cancers experience persistent ER stress, leading to hyperactive IRE1 signaling that limits tumor control. However, amplifying the adaptive arm of the IRE1 UPR serves to eliminate mitochondrial toxicity and protect T cells from chronic ER stress, yielding robust tumor engraftment and long-term tumor immunity. Our findings establish the UPRs essential protective role in antitumor immunity.

immunology↗

bioRxiv2021

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

Cell cycle gene expression programs fuel proliferation and are dysregulated in many cancers. The retinoblastoma-family proteins, RB, p130/RBL2 and p107/RBL1, coordinately repress cell cycle gene expression, inhibiting proliferation and suppressing tumorigenesis. Ubiquitin-dependent protein degradation is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of several hundred CRISPR/Cas9 loss-of-function screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a non-degradable p130 represses cell cycle gene expression and strongly reduces proliferation. These data suggest that SCFcyclin F plays a key role in the CDK-RB network and raises the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

cell biology↗

bioRxiv2020

In silico APC/C substrate discovery reveals cell cycle degradation of chromatin regulators including UHRF1

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates and we show that several chromatin proteins bind APC/C, oscillate during the cell cycle and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

cell biology↗

bioRxiv2019

Cell cycle oscillators underlying orderly proteolysis of E2F8

E2F8 is a transcriptional repressor that antagonizes the canonical cell cycle transcription factor E2F1. Despite the importance of this atypical E2F family member in cell cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control its dynamics. To address this question, we developed a complementary set of static and dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, and a full transition from pro-metaphase to G1. This revealed an interlocking molecular switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 sites in E2F8 and the activation of APC/CCdh1, but not APC/CCdc20. Further, we revealed a differential stability of E2F8, accounting for its accumulation in late G1 while APC/CCdh1 is still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in promoting orderly cell cycle progression.

cell biology↗