Biology subjects

Zambo, B.

Publications and source records attributed to Zambo, B..

11 recordsLinked to original sources

bioRxiv2026

An NR2F1-dependent retinoic acid network controls retinal specialization in mice and reveals foveal hypoplasia in patients with BBSOAS

The molecular programs that establish specialized retinal regions during development are essential for high-acuity vision, yet how their disruption contributes to human visual disorders remains poorly understood. Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), caused by pathogenic variants in NR2F1 and characterized by visual impairment, provides an opportunity to investigate these mechanisms. Using single-cell RNA sequencing of three complementary Nr2f1 mouse models, including two carrying patient-specific mutations, we identified a shared Nr2f1-dependent transcriptional program enriched in retinoic acid (RA) pathway genes. Loss or mutation of Nr2f1 disrupted the spatial organization of RA signaling, most prominently by expanding the dorso-equatorial Cyp26a1 expression domain into ventral retina and reducing ventral determinants such as Vax2. These molecular changes were associated with altered dorso-ventral distribution of S- and M-opsin-expressing cone photoreceptors. We further demonstrate that human NR2F1 binds a conserved regulatory region upstream of CYP26A1, supporting its direct role in regulating local RA availability. Finally, high-resolution optical coherence tomography in individuals with BBSOAS revealed reproducible foveal abnormalities, including a smaller and shallower foveal pit and increased central retinal thickness, consistent with foveal hypoplasia. These findings uncover a previously unrecognized retina-intrinsic component of BBSOAS visual pathology and establish an NR2F1-RA/CYP26A1 regulatory axis linking developmental retinal regionalization to human foveal specialization.

developmental biology↗

bioRxiv2026

FMRP controls diacylglycerol-dependent neuronal activity by mediating m6A-regulated translation efficiency

Fragile X syndrome (FXS), a leading inherited cause of intellectual developmental disorder and autism, results from loss of the RNA-binding protein FMRP. Loss of FMRP causes excessive neuronal protein synthesis contributing to widespread functional disturbances, yet the mechanisms linking FMRP to specific mRNA targets remain unclear. We show that FMRP promotes translation of the brain mRNA DGK{kappa} by binding mA-modified repetitive RNA motifs, thereby relieving a translational block encoded within its sequence. FMRP loss sharply reduces neuronal DGK{kappa}, and DGK{kappa} depletion alone reproduces hallmark FXS phenotypes, including hyperactivity, compulsive behavior, overgrowth, dendritic spine abnormalities, overactivated diacylglycerol signaling and increased protein synthesis. These findings identify DGK{kappa} as a key effector of FMRP function and establish a regulatory axis where mA RNA modification modulates neuronal translation. This work defines new principles of translational control in neurodevelopmental disorders and positions DGK{kappa} as a central driver of FXS pathogenesis.

molecular biology↗

bioRxiv2025

Structural analysis of HERC2/UBE3A and HERC2/DOCK10 complexes provides new insights into the molecular basis of Angelman, Angelman-like and Dup15q Syndromes

UBE3A and HERC2 are two mutually interacting HECT E3 Ubiquitin ligases whose genes are altered in 15q11.2-13.1 Duplication (Dup15q) Syndrome, Angelman Syndrome (AS) and other phenotypically-related mental retardation syndromes. Using quantitative binding assays, X-ray crystallography and sequence conservation analysis, we show that the HERC2/UBE3A complex occurs in probably most animals with a central nervous system, via a conserved interface involving the RLD2 domain of HERC2 and a "DxDKDxD" motif of UBE3A. We found that HERC2 also recognizes and binds to similar DxDKDxD motifs within a handful of other proteins relevant to brain development (DOCK10, PCM1, USP35, BAZ2B, ARID4A, ARIP4, RERE and MYT1). We further investigated the interaction of HERC2 with DOCK10, a RAC1- and CDC42-GEF protein that regulates dendritic spine morphogenesis in hippocampal neurons. Both disruption of the HERC2-binding motif in DOCK10 and knockdown of HERC2 affected the GEF activity of DOCK10. We also show that the DOCK10-induced dendritic spine formation is dependent on its ability to bind HERC2. Structural modeling of full-length DOCK10, free or bound to either RLD2, CDC42 or RAC1 indicates that the GEF activation of full-length DOCK10 requires a conformational change that is stimulated by binding to HERC2. Based on our data, we propose that under pathological conditions, in developing brains with an abnormal dosage of either HERC2 or its dominant partner UBE3A, increased or decreased amounts of HERC2/DOCK10 complexes could lead to altered GTPase activation. This in turn could affect dendritic spine formation and neurodevelopment.

biochemistry↗

bioRxiv2025

The non-catalytic ϵ DNA polymerase subunit is an NPF motif recognition protein

Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase {varepsilon} (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analysis show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affinity screens identify NPF-containing nuclear proteins (e.g., WDHD1, DONSON, TTF2) that bind POLE2 with micromolar affinities, and their motif mutations abolish binding in cell extracts. Although POLE2 primarily tethers the catalytic POLE subunit to replication forks, these results indicate that it can also recruit various NPF-bearing partners involved in replication, DNA repair, and transcription regulation. Notably, NPF motifs optimized for EH-domain binding can still associate with POLE2, highlighting the inherent degeneracy of SLiM-mediated networks. Overall, these findings establish POLE2 as a central hub linking replication with other processes via broad NPF-motif recognition.

biochemistry↗

bioRxiv2024

Identification and quantitative characterization of protein-DNA interactions with the native holdup assay: GATA1 regulation of the plasma membrane calcium pump

GATA1 is a crucial transcription factor involved in hematopoiesis, and mutations in this gene are linked to severe hematological disorders, including anemia, thrombocytopenia, Down-syndrome related transient abnormal myelopoiesis (DS-TAM) and acute megakaryocytic leukemia (DS- AMKL). Despite significant clinical interest in the molecular level characterization of GATA1 mutations, a comprehensive understanding of their impact on DNA binding is limited. Efforts to conduct detailed studies on full-length recombinant GATA1 have faced significant technical challenges, while alternative approaches are limited by low throughput or qualitative nature. Here, we introduce a native holdup (nHU) assay designed to systematically quantify DNA-protein interactions and suitable for studying the impact of transcription factor mutations on DNA-binding affinity. First, we demonstrate, using the erythroid-specific ATP2B4 promoter as a model, that nHU can capture sequence-specific interactions and detect even subtle differences in DNA binding affinities of GATA1. Then, we quantitatively characterize the impact of pathological mutations on DNA binding affinities in the context of full-length human GATA1. Our findings reveal that the short isoform, lacking the N-terminal transactivation domain (N-TAD), binds to DNA with increased affinity, while the R307C mutation reduces binding to the ATP2B4 erythroid promoter. In harmony with these observations, GATA1s exhibits increased functional activity, while the R307C mutation results in decreased activity. This study demonstrates the power of the nHU assay for studying DNA interactions of transcription factor variants and providing insight into the molecular mechanism of related diseases.

molecular biology↗

bioRxiv2023

Comparative analysis of PDZ-binding motifs in the diacylglycerol kinase family

Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in cells. Certain DGK enzymes possess C-terminal sequences that encode potential PDZ-binding motifs (PBMs), which could be involved in their recruitment into supramolecular signaling complexes. In this study, we used two different interactomic approaches, quantitative native holdup (nHU) and qualitative affinity purification (AP), both coupled to mass spectrometry (MS) to investigate the PDZ partners associated with the potential PBMs of DGKs. Complementing these results with site-specific affinity interactomic data measured on isolated PDZ domain fragments and PBM motifs, as well as evolutionary conservation analysis of the PBMs of DGKs, we explored functional differences within different DGK groups. All our results indicate that putative PBM sequences of type II enzymes are likely to be nonfunctional. In contrast, type IV enzymes possess highly promiscuous PBMs that interact with a set of PDZ proteins with very similar affinity interactomes. The combination of various interactomic assays and evolutionary analyses provides a useful strategy for identifying functional domains and motifs within diverse enzyme families.

molecular biology↗

bioRxiv2023

Affinity-ranking BIN1 interactions and screening natural BIN1 variants that may cause neuromuscular disorders

Truncation of the protein-protein interaction SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1, Amphiphysin 2) protein leads to centronuclear myopathy. Here, we assessed the impact of a set of naturally observed, previously uncharacterized BIN1 SH3 domain variants using conventional in vitro and cell-based assays monitoring the BIN1 interaction with dynamin 2 (DNM2) and identified potentially harmful ones that can be also tentatively connected to neuromuscular disorders. However, SH3 domains are typically promiscuous and it is expected that other, so-far unknown partners of BIN1 exist besides DNM2, that also participate in the development of centronuclear myopathy. In order to shed light on these other relevant interaction partners and to get a holistic picture of the pathomechanism behind BIN1 SH3 domain variants, we used affinity interactomics. We identified hundreds of new BIN1 interaction partners proteome-wide, among which many appear to participate in cell division, suggesting a critical role of BIN1 in the regulation of mitosis. Finally, we show that the identified BIN1 mutations indeed cause proteome-wide affinity perturbation, signifying the importance of employing unbiased affinity interactomic approaches.

biochemistry↗

bioRxiv2022

Native holdup (nHU) to measure binding affinities from cell extracts

Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

systems biology↗

bioRxiv2021

Quantitative fragmentomics allow affinity mapping of interactomes

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here we measured the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complement protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within realistic reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus (HPV) E6 oncoprotein deeply impacts the host cell proteome way beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

systems biology↗

bioRxiv2021

AAV-delivery of diacylglycerol kinase kappa achieves long-term rescue of Fmr1-KO mouse model deficits of fragile X syndrome

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. It results from the lack of the RNA binding protein FMRP and is associated with the overactivation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk) is a main mRNA target of FMRP in cortical neurons. Here we show that diacylglycerol kinase kappa (DGKk), when modified as to become FMRP-independent and delivered into the brain of adolescent mice using adeno-associated viral vectors, corrects brain diacylglycerol and phosphatidic acid homeostasis and the main phenotypic behaviors of the Fmr1-KO mouse model of FXS. Thus, DGKk appears as a key triggering factor of FXS pathomechanism while providing a possible means of intervention for FXS gene therapy. One sentence summaryDGKk gene therapy in Fmr1-KO mouse model

neuroscience↗

bioRxiv2020

Pioglitazone improves deficits of Fmr1-KO mouse model of Fragile X syndrome by interfering with excessive diacylglycerol signaling

Fragile X syndrome (FXS), the leading cause of familial intellectual disability, is an uncured disease caused by the absence or loss of function of the FMRP protein. FMRP is an RNA binding protein that controls the translation of specific proteins in neurons. A main target of FMRP in neurons is diacylglycerol kinase kappa (DGKk) and the loss of FMRP leads to a loss of DGK activity causing a diacylglycerol excess in the brain. Excessive diacylglycerol signaling could be a significant contributor to the pathomechanism of FXS. Here we tested the contribution of DAG-signaling in Fmr1-KO mouse model of FXS and we show that pioglitazone, a widely prescribed drug for type 2 diabetes, has ability to correct excessive DAG signaling in the brain and rescue behavioral alterations of the Fmr1-KO mouse. This study highlights the role of lipid signaling homeostasis in FXS and provides arguments to support the testing of pioglitazone for treatment of FXS.

neuroscience↗