Biology subjects

Pons, M.

Publications and source records attributed to Pons, M..

11 recordsLinked to original sources

bioRxiv2025

The Deacetylases HDAC1 and HDAC2 Safeguard BCR-ABL-positive Cells from Replication Stress-Induced Apoptosis via the Nuclear to Mitochondrial p73-NOXA Axis

Histone deacetylases (HDACs) are key epigenetic regulators that are frequently dysregulated in cancer cells. Context-specific dependencies of tumor cell survival on HDACs and the mechanisms through which HDACs determine cell stress responses by specific oncogenic pathways remain to be understood. Here we unravel how the class I deacetylases HDAC1 and HDAC2 control the fate of chronic myeloid leukemia (CML) cells undergoing DNA replication stress. Compared to normal myeloid cells (n=690), CML cells overexpress HDAC1 and HDAC2 (n=234-274). We reveal that HDAC1 and HDAC2 protect cultured and primary CML cells with the hyperactive BCR-ABL tyrosine kinase from programmed cell death through apoptosis upon DNA replication stress. Using transcriptomics and genetically defined knockdown and knockout model systems, we demonstrate that these effects depend on the transcription factor p73 and its pro-apoptotic target gene NOXA. Upon DNA replication stress, p73 binds to the NOXA promoter but only upon additional inactivation of HDAC1/HDAC2 the NOXA gene becomes transcribed. BCR-ABL translocate to the nucleus to catalyze p73 phosphorylation at tyrosine-99 for the induction of p73 and mitochondrial NOXA. Thus, the BCR-ABL oncogene creates a selective vulnerability to HDAC1/HDAC2 inhibitors, driving a cytotoxic shift in replication stress responses towards apoptosis. These data highlight HDAC1/HDAC2 as potential therapeutic targets in CML. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/694110v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1ec0c51org.highwire.dtl.DTLVardef@1cd759org.highwire.dtl.DTLVardef@17e29a8org.highwire.dtl.DTLVardef@1e0fb50_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Lipid-driven SRC self-association modulates its transformation capacity

Src tyrosine kinase is anchored to the plasma membrane, however, the contribution of membrane lipids to its regulation remains elusive. Here we report Src self-association through a lysine cluster in the Src SH4 region mediated by lipids in human cells and in vitro. Mutation of the lysine cluster to arginine modulates Src self-association and its transforming function in human cells. This mechanism may also apply to other membrane-associated signaling proteins that have similar lysine clusters in their unstructured regions. Lipid-anchored micron-sized condensates of full length Src are formed in supported homogeneous lipid bilayers (i.e independently of lipid phase separation). Condensates are also formed by the purified Src N-terminal regulatory element, including the myristoylated SH4 domain, the intrinsically disordered Unique domain and the globular SH3 domain. The isolated SH4 domain forms small protein-lipid clusters, but not micron-size condensates. Our findings reveal lipid-mediated kinase self-association as an additional mechanism for Src regulation. Significance StatementCondensate formation, often associated to intrinsically disordered protein regions is emerging as a widespread regulatory mechanism. Protein phase separation has been linked to the regulation of kinases recruited to condensates formed by other proteins. Here we show i) that the N-terminal disordered region of Src kinase drives lipid-mediated self-association to form condensates on the cell surface, ii) a lysine cluster in the N-terminal SH4 domain is essential for this process, iii) mutation of these lysine residues to arginine increases self-association in vitro and in vivo, and iv) self-association of Src results in enhanced transforming capacity in human cells. Similar lysine clusters are present in juxtamembrane regions suggest a general mechanism for modulating protein-protein interactions by lipids. The article shows the formation of condensates by Src on supported lipid membranes mediated by lipids, dependent of a conserved lysine cluster in its SH4 domain, and modulating Srcs transforming capacity. O_LIFull length Src and truncated variants including its myristoylated SH4 domain self-associate on the surface of homogeneous lipid bilayers. C_LIO_LIThe interaction of a lysine cluster in SH4 domain with lipids modulates Src self-association C_LIO_LIAtomic force microscopy shows the formation of micron-size condensates and smaller clusters on the surface of supported lipid bilayers, depending on the lipid composition or the presence of mutations in the lysine cluster C_LIO_LISrc self-association is confirmed in human cells and the Src transforming function of Src in human cells is modulated by lipid-mediated self-association C_LI

molecular biology↗

bioRxiv2024

Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasome-dependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.

microbiology↗

bioRxiv2023

Modulation of functional phosphorylation sites by basic residues in the Unique domain of c-Src

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulate the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The preexisting positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or coupling various phosphosites into a functional unit. In this paper we use pH dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight on the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationship observed between the mutation induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH induced chemical shifts of the NH groups of these residues provides a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

biophysics↗

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗

bioRxiv2022

Sea lamprey nests promote the diversity of benthic macroinvertebrate assemblages

The habitat heterogeneity hypothesis states that increased habitat heterogeneity promotes species diversity through increased availability of ecological niches. We aimed at describing the local-scale effects of nests of the sea lamprey (Petromyzon marinus L.) as ecosystem engineer on macroinvertebrate assemblages. We hypothesized that increased streambed physical heterogeneity caused by sea lamprey spawning would modify invertebrate assemblages and specific biologic traits and promote reach-scale diversity. We sampled thirty lamprey nests of the Nive River in three zones: the unmodified riverbed (control) and zones corresponding to the nest: the area excavated (pit) and the downstream accumulation of pebbles and cobbles (mound). The increased habitat heterogeneity created by lamprey was traduced by biological heterogeneity with a reduced density of invertebrates (1160 to 6540 individuals per m2 in control, 680 to 6460 individuals per m2 in pit and 1980 to 6240 individuals per m2 in mound) and number of taxa (23.5 {+/-} 3.9 taxa for control, 18.6 {+/-} 3.9 taxa in pit and 21.2 {+/-} 4.5 taxa for mound) in the pit compared to other zones. However the overall taxa diversity in nest increased with 82 {+/-} 14 taxa compared to the 69 {+/-} 8 taxa estimated in control zone. Diversity indices were consistent with the previous results indicating a loss of diversity in pit but a higher {beta} diversity between a pit and a mound than between two control zones, especially considering Morisita index accounting for taxa abundance. Trait analysis showed high functional diversity within zones with a reduced proportion of collectors, scrapers, shredders, litter/mud preference and small invertebrates in mound, while the proportion of "slabs, blocks, stones and pebbles" preference and largest invertebrates increased. Pit presented the opposite trend, while control had globally intermediate trait proportions. Our results highlight important effects on species and functional diversity due to habitat heterogeneity created by a nest-building species, what can ultimately influence food webs and nutrient processes in river ecosystems.

ecology↗

bioRxiv2022

Ligands tune the local and global motions of neurotensin receptor 1 (NTS1): a DFT-guided solution NMR analysis

Unlike many signaling proteins that function as binary switches between on and off states, G protein-coupled receptors (GPCRs) exhibit basal activity that can be increased or decreased by numerous ligands. A given receptor can recognize multiple ligands, allosteric modulators, and transducers to create a complex free energy landscape. Many of the lowest energy states have been captured by static structural techniques while detailing the wells widths, metastable states, and the transition between them, is still in its infancy. Nuclear magnetic resonance (NMR) spectroscopy can monitor the structure and dynamics of GPCR ensembles across fifteen orders-of-magnitude, but technical challenges have limited its application to super-microsecond timescales. Focusing on a prototypical peptide-binding GPCR, the neurotensin receptor 1 (NTS1), we employed NMR and density functional theory (DFT) to probe global sub-nanosecond motions. The near random coil chemical shifts of the apo receptor produced a poor correlation with theoretical predictions that may indicate a high degree of conformational averaging in solution, a crystallization artifact, or both. Whereas orthosteric agonists and antagonists both rigidified the receptor, but to varying degrees, which suggests conformational entropy differentially contributes to their respective pharmacology. The strong correlations of observed and theoretical chemical shifts lend confidence to interpreting spectra in terms of local structure, methyl dihedral angle geometry, and pico-second timescale transitions. Together, our results suggest a role for sub-nanosecond dynamics and conformational entropy in GPCR ligand discrimination.

biophysics↗

bioRxiv2022

Lipid-mediated dimerization of membrane-anchored c-Src is driven by a cluster of lysine residues in the N-terminal SH4 domain.

The membrane-anchored c-Src tyrosine kinase mediates signaling from a wide range of cell surface receptors controlling cell growth, adhesion, and survival. c-Src deregulation is associated with cancer. Dimerization appears to be a novel layer of regulation through a yet unclear mechanism. Binding of c-Src tyrosine kinase to the plasma membrane is mediated by the myristoylated and strongly positively charged N-terminal SH4 domain. Although activation of c-Src is known to require phosphorylation by a second c-Src molecule, electrostatic repulsion between the charged residues was considered to prevent dimerization. Here we show that a cluster of positively charged lysine residues in c-Src SH4 domain not only does not prevent dimerization but, in fact, enhances it through a lipid-mediated process. Dimerization not only depends on the number of positive charges but also on their position and the nature of the charged residues. Replacement of lysine by arginine increases dimerization in vitro and in vivo and, in HEK293T cells, causes a two-fold increase in tyrosine phosphorylation. Lipid mediated protein-protein interactions induced by clusters of basic residues may represent a general mechanism for modulating cell signaling, consistent with the abundance of positively charged residues in the juxta membrane region of many signaling proteins.

biophysics↗

bioRxiv2021

NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The pro-apoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models we identified an inhibitor of the transcription factor heterodimer CBF{beta}/RUNX1. By genetic gain and loss of function experiments we validated that the mode of action depends on RUNX1 and NOXA. Of note, RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a novel way to target a therapy resistant PDAC, an unmet clinical need. SignificanceRecent evidence demonstrated the existence of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), which resist all current therapies. The paucity of therapeutic options, including a complete lack of targeted therapies, underscore the urgent and unmet medical need for the identification of targets and novel treatment strategies for PDAC. Our study unravels a function of the transcription factor RUNX1 in apoptosis regulation in PDAC. We show that pharmacological RUNX1 inhibition in PDAC is feasible and leads to NOXA-dependent apoptosis. The development of targeted therapies that influence the transcriptional landscape of PDAC might have great benefits for patients who are resistant to conventional therapies. RUNX1 Inhibition as a new therapeutic intervention offers an attractive strategy for future therapies.

cancer biology↗

bioRxiv2021

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

The membrane anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Srcs oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

cancer biology↗

bioRxiv2018

The systemic activin response to pancreatic cancer: Implications for effective cancer cachexia therapy

Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy with high rates of cachexia. Serum activin correlates with PDAC cachexia and mortality, while activin administration causes cachexia in mice. We studied activin in human tumors and in mice with orthotopic or genetic PDAC. Cachexia severity correlated with activin expression in tumor lines. Activins were expressed in both cancer and tumor stromal cells, but also in organs in murine PDAC cachexia. Tumor cells expressed activin-{beta}A, or Inhba, while organs expressed both activin-{beta}A and activin-{beta}B, or Inhbb. PDAC elicits activin expression; PDAC conditioned medium induced activin and atrophy of myotubes. Treatment with the activin trap, ACVR2B/Fc, reduced cachexia and prolonged survival in mice with activin-low tumors, and reduced cachexia in activin-high tumors, without affecting activin expression in organs. Mice expressing dominant negative ACVR2B in muscle were protected for weight loss but not survival. Overall our results indicate that PDAC induces a systemic activin response, leading to cachexia, and that activin targets might include organs beyond muscle. Targeting of both tumor-derived and host-derived activins could improve cachexia therapy.

cancer biology↗