Biology subjects

Zarzuela, E.

Publications and source records attributed to Zarzuela, E..

11 recordsLinked to original sources

bioRxiv2026

The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism

RAF kinases activate MEK in the RAS-MAPK signaling pathway, and changes in RAF kinase signaling have been linked to tumor formation. RAF1 requires the HSP90-CDC37 chaperone system for proper activation, but how the HSP90-CDC37 chaperone system regulates RAF kinase maturation remains enigmatic. We present novel cryo-EM structures of previously uncharacterized RAF1 chaperone complexes, including a 2:2:2 RAF1-HSP90-CDC37 complex (RRHCC), intermediate assemblies (RHCC), and a RAF1-HSP90-CDC37-p23 complex (RHCp23). These reveal an asymmetric stepwise folding mechanism unique among HSP90 kinase clients in which one RAF1 threads through the HSP90 lumen while another is captured in a "casting mold" formed by CDC37 and HSP90 that stabilizes the partially folded C helix of RAF1. The RHCp23 structure shows how p23 cooperates with CDC37 to regulate ATP hydrolysis and client release. The HSP90-CDC37 system supports pre-dimerization of RAF1 and BRAFV600E homodimers and RAF1 heterodimers, a mechanism unique to RAF among kinase clients of HSP90. Phosphoproteomics reveals selective activating phosphorylations within RRHCC. These RAF isoform complexes differentially activate MEK signaling and cell proliferation, establishing HSP90-CDC37 as not just a passive stabilizer but an active regulator of RAF signaling with therapeutic implications.

biochemistry↗

bioRxiv2025

Cyclin Y Overexpression Drives a Fatal Metabolic Syndrome via Defective Glucose Homeostasis

Cyclin Y (CCNY) is a membrane-associated, non-canonical cyclin best known for regulating WNT/beta-catenin signaling via the recruitment of CDK14/16 protein kinases. Whereas its role in the activation of members of the atypical CDK14-18 subfamily is established, its function in systemic metabolism remains poorly defined. Here, we report that CCNY overexpression drives a fatal metabolic syndrome. Using a novel inducible knock-in mouse model, we demonstrate that CCNY overexpression causes severe cachexia and profound hypoglycemia, resulting in death of adult mice independent of tumor burden. While these mutant mice maintain normal food intake and hepatic synthetic function, they succumb to metabolic starving state. Proteomics reveals that CCNY interacts with and stabilizes Pyruvate Dehydrogenase Kinase 4 (PDK4), in agreement with defective pyruvate use at mitochondria and enforcing a Warburg-like shift to aerobic glycolysis. Phosphoproteomic analysis indicates activation of apoptotic pathways and defective phosphorylation of several enzymes critical for glycolysis and gluconeogenesis as well as amino acid metabolism in addition to other metabolic routes. Altogether, these data suggest that CCNY overexpression uncouples nutrient sensing from utilization, a finding with possible therapeutic implications in CCNY-high cancers with similar changes in metabolic pathways.

cell biology↗

bioRxiv2025

CDC14 phosphatases control adipogenesis via PPARγ de-phosphorylation

Adipogenesis is a finely tuned process requiring an appropriate balance between proliferation and differentiation. We demonstrate that CDC14, a CDK-counteracting phosphatase, regulates adipogenesis and glucose metabolism in mammals. Simultaneous depletion of CDC14A and CDC14B protects against high-fat diet-induced obesity, hepatic steatosis and glucose intolerance in vivo. Lack of these phosphatases blocks differentiation of stem cells into adipocytes, a defect rescued by the PPAR{gamma} agonist troglitazone. Mechanistically, we show that CDC14 counteracts ERK2 and CDK5-induced phosphorylation of PPAR{gamma}, a master regulator of adipogenesis, by directly dephosphorylating S112, S273 and T296 residues. This study supports the idea that cell cycle regulators in multicellular organisms evolved to coordinate the balance between proliferation and differentiation in different cell types. Furthermore, since PPAR{gamma} is a key target of anti-diabetic drugs and a central regulator of adipogenesis and metabolism, these findings may have important implications for treatment of metabolic diseases such as type 2 diabetes.

cell biology↗

bioRxiv2025

STAG2 cohesin cooperates with DREAM to maintain quiescence and suppress tumourigenesis in the urothelium

The maintenance of quiescence is essential for tissue homeostasis. STAG2 is one of the few genes mutated in the normal urothelium of organ donors, with mutant cells undergoing positive selection 1. STAG2 is also a major tumour suppressor gene 2-4 and its inactivation is an early event in bladder carcinogenesis 1,3. However, how STAG2, a cohesin component, regulates urothelial homeostasis remains largely unknown. Here, we demonstrate that Stag2 inactivation in normal murine urothelial cells interferes with differentiation programs, triggers transient cell cycle entry, and primes cells for clonal expansion under stress. Moreover, STAG2 loss enhances tumor formation in urothelial cells expressing mutant FGFR3 - the key oncogene in bladder cancer 5. We reveal that STAG2 cooperates with the DREAM transcriptional complex, a master regulator of quiescence 6,7, by binding to shared genomic sites, including cell cycle control genes. STAG2 loss alters DREAM target expression, complex composition, and chromatin distribution, and leads to rewiring of chromatin interactions involving DREAM binding motifs in genes critical for cell cycle entry. Our findings provide compelling evidence that STAG2 loss disrupts in 3D genome organization through a novel mechanism involving the DREAM complex, thereby impairing homeostatic quiescence and increasing oncogenic sensitivity.

cancer biology↗

bioRxiv2025

The tyrosine kinase inhibitor GNF-7 targets senescent cells through allosteric activation of GCN2.

The one-two-punch approach refers to the sequential administration of two different chemotherapies, the second of which targets cancer cells that resisted the initial treatment. To find such a second punch, we performed a chemical screen to find drugs that are preferentially toxic for cells with an activated DNA damage response (DDR). This screen identified the tyrosine kinase inhibitor GNF-7 as a top hit. Subsequent work revealed that GNF-7 is a potent senolytic, even when senescence is triggered by therapies that do not activate the DDR. Consistently, GNF-7 is highly efficacious to kill cancer cells previously treated with CDK4/6 inhibitors, including in patient-derived organoids and mouse xenografts. Surprisingly, the senolytic effect of GNF-7 is not mediated by the inhibition of a tyrosine kinase (TK), but rather by the activation of GCN2, an effect previously reported for other TK inhibitors. Together, our study reports the discovery of a novel senolytic agent that strongly synergizes with CDK4/6 inhibitors when applied sequentially and expands our understanding of the mechanisms behind the anticancer effects of TK inhibitors.

cancer biology↗

bioRxiv2025

The oncogenic CCDC6-RET fusion product is a dual ATP and ADP-dependent kinase that functions via cis-phosphorylation

Gene fusions products involving protein kinases are known drivers in human cancers and actionable targets for personalized therapy, yet the structural and molecular determinants that control their function are largely unexplored. Here we show that a CCDC6-RET fusion product, a driver and therapeutic target in lung and thyroid cancers, is a highly active dimeric kinase in solution. Time-resolved mass spectrometry analysis together with a robust biochemical and biophysical characterization reveal that CCDC6-RET functions as a dual ATP- and ADP-dependent kinase able to bind both nucleotides and uses them as phosphoryl donors. We also identify a crosstalk between the c-terminal and the activation segments, uncovering a mutually exclusive dependency by the former on activation loop phospho-sites controlling both the processing and the catalytic activity of the fusion protein. Furthermore, we generated a 3D-assembly of a CCDC6-RET homodimer combining electron microscopy (EM) single particle, small-angle X-ray scattering (SAXS) and in silico structural analyses. Our structural model together with cross-linking mass spectrometry data demonstrated that CCDC6-RET forms a face-to-face trans-inhibited dimer in the apo state characterized by intermolecular-crosslinked activation segments. Upon nucleotide binding and catalytic domains reorientation, fast activation loop phosphorylation is driven by a mechanism in cis. Our work uncover for the first time the molecular and structural determinants that controls CCDC6-RET function and provides a solid framework to study the structure and function of other RET fusion products.

biochemistry↗

bioRxiv2022

Peroxiredoxin 2 is required for the redox mediated adaptation to exercise

Exercise generates a site-specific increase in Reactive Oxygen Species (ROS) within muscle required for a beneficial adaptive response by activation of specific signalling pathways. Here, we demonstrate that Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxiredoxin, is required for the adaptive beneficial hormesis response to H2O2. A short bolus addition of H2O2 increases mitochondrial capacity and improves myogenesis of cultured myoblasts, this beneficial adaptive response was suppressed in myoblasts with decreased expression of cytoplasmic Prdxs. A swimming exercise protocol in C. elegans increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. In contrast, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise identified distinct changes in the proteome of N2 and prdx-2 mutants. Furthermore, a redox proteomic approach to quantify reversible oxidation of individual Cysteine residues revealed a relatively more oxidised redox state following exercise in the prdx-2 mutants. Our results demonstrate that conserved cytoplasmic 2-Cys Peroxiredoxins are required for the beneficial adaptive response to a physiological stress.

molecular biology↗

bioRxiv2022

Induction of senescence renders cancer cells highly immunogenic

Cellular senescence is a stress response that activates innate immunity. However, the interplay between senescent cells and the adaptive immune system remains largely unexplored. Here, we show that senescent cells display enhanced MHC class I (MHC-I) antigen processing and presentation. Immunization of mice with senescent syngeneic fibroblasts generates CD8 T cells reactive against both normal and senescent fibroblasts, some of them targeting senescence-associated MHC-I-peptides. In the context of cancer, we demonstrate that senescent cancer cells trigger strong anti-tumor protection mediated by antigen-presenting cells and CD8 T cells. This response is superior to the protection elicited by cells undergoing immunogenic cell death. Finally, induction of senescence in patient-derived cancer cells exacerbates the activation of autologous tumor-reactive CD8 tumor-infiltrating lymphocytes (TILs) with no effect on non-reactive TILs. Our study indicates that immunization with senescent cancer cells strongly activates anti-tumor immunity, and this can be exploited for cancer therapy. STATEMENT OF SIGNIFICANCEOur study shows that senescent cells are endowed with a high immunogenic potential, superior to the gold standard of immunogenic cell death. The induction of senescence in cancer cells can be exploited to develop efficient and protective CD8-dependent anti-tumor immune responses.

cancer biology↗

bioRxiv2022

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation

RAF kinases are RAS-activated enzymes that initiate signalling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryo-electron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities to attempt the pharmacological degradation of RAF1 for therapeutic purposes.

biochemistry↗

bioRxiv2020

Wnt-mediated interactions of tumor-initiating cells with a macrophage niche drive skin tumor formation

Skin tumor-initiating stem cells (tSCs) fuel skin squamous cell carcinoma (SCC) formation and development in a complex tumor microenvironment, but a role for immune cells in the tSC niche governing the process of tumor formation has remained elusive. Here, we define the existence of a tSC-macrophage niche, expressing high levels of Wnt ligands. Using conditional mouse genetic models to abrogate the secretion of Wnts, we show that both hair follicle SC- and macrophage-derived Wnts are essential for driving skin tumorigenesis, tSCs maintenance, and counteracting tumor regression. Loss of Wnts in either population uncouples the tSC-macrophage association. The proteomic signature of SCC cells reveals CD99 as a Wnt-dependent receptor for the macrophagecancer cell interaction. These results establish a role for a macrophage-tSC niche in governing SCC initiation and maintenance, uncovering potential candidates for immunoprevention against SCC. One Sentence SummaryA macrophage-skin tumor-initiating cells bonding Wnt loop drives tumorigenesis.

cancer biology↗

bioRxiv2018

DNA and RNA binding mediate the toxicity of arginine-rich peptides encoded by C9ORF72 GGGGCC repeats

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

neuroscience↗