Molecular Switch Research Articles

Phytohormones and related genes function as physiological and molecular switches regulating water stress response in the sunflower.

The online version contains supplementary material available at 10.1007/s12298-024-01497-8.

Abstract Tu126: Unveiling the Role of GRAF1 in Orchestrating Mitophagy and Metabolic Flexibility in Stressed Cardiomyocytes

Mitochondria are essential for cardiomyocyte contraction by generating ATP and orchestrating metabolic pathways. Understanding how mitophagy—selective removal of damaged mitochondria in lysosome—and metabolic flexibility are coordinated is crucial for cardiac function under physiological and pathological conditions. Our study explores the molecular mechanisms behind this coordination in cardiomyocytes. We identify GRAF1(Arhgap26) as a pivotal mediator in PINK1-Parkin dependent mitophagy, facilitating damaged mitochondria clearance and regulating mitochondrial substrate flexibility. Depletion of GRAF1 in neonatal rat ventricular cardiomyocytes (NRVCMs) compromises mitochondrial function, evidenced by reduced membrane potential, impaired oxidative phosphorylation, and elevated cleaved Caspase 3 levels. Moreover, GRAF1 depletion attenuates LC3II activation and increases mitochondrial mass following toxin-induced stress. Using tamoxifen-inducible cardiomyocyte-restricted GRAF1 knockout mice (GRAF1 CKO ), we observe normal cardiac function under basal conditions. However, upon isoproterenol (ISO) treatment, GRAF1 CKO mice display cardiac dysfunction and reduced mitophagy. Metabolomics analysis reveal distinct metabolic signatures between ISO-treated control and GRAF1 CKO mice, with impaired fuel flexibility observed in GRAF1 CKO mice, as highlighted by impaired glucose utilization relative to control mice. Mechanistically, phosphorylation of specific sites (S668, T670, and S671) within the proline-rich region of GRAF1 by PINK1 or PINK1-dependent kinases regulates its dual functions. This phosphorylation event releases the autoinhibitory state of the SH3 domain, enabling it to interact with ABI2 and WAVE2 complex for actin remodeling necessary for efficient mitochondria clearance. Additionally, phosphorylation enhances GRAF1’s affinity for Malonyl-CoA Decarboxylase (MCD), promoting MCD degradation and subsequent elevation of cellular malonyl-CoA levels. This rise in malonyl-CoA inhibits carnitine pamitoyl transferase-I (CPT-I), thereby suppressing fatty acid oxidation while promoting glucose oxidation in mitochondria, contributing to the mitochondrial metabolic flexibility. In summary, our findings elucidate GRAF1's role in orchestrating mitophagy and metabolic flexibility in stressed cardiomyocytes. GRAF1 phosphorylation serves as a molecular switch, ensuring efficient mitochondrial clearance and substrate utilization to maintain cardiac energetics and function.

Advances in functional studies of plant MYC transcription factors.

Myelocytomatosis (MYC) transcription factors (TFs) belong to the basic helix-loop-helix (bHLH) family in plants and play a central role in governing a wide range of physiological processes. These processes encompass plant growth, development, adaptation to biotic and abiotic stresses, as well as secondary metabolism. In recent decades, significant strides have been made in comprehending the multifaceted regulatory functions of MYCs. This advancement has been achieved through the cloning of MYCs and the characterization of plants with MYC deficiencies or overexpression, employing comprehensive genome-wide 'omics' and protein-protein interaction technologies. MYCs act as pivotal components in integrating signals from various phytohormones' transcriptional regulators to orchestrate genome-wide transcriptional reprogramming. In this review, we have compiled current research on the role of MYCs as molecular switches that modulate signal transduction pathways mediated by phytohormones and phytochromes. This comprehensive overview allows us to address lingering questions regarding the interplay of signals in response to environmental cues and developmental shift. It also sheds light on the potential implications for enhancing plant resistance to diverse biotic and abiotic stresses through genetic improvements achieved by plant breeding and synthetic biology efforts.

Neurobiochemical Disturbances in Psychosis and their Implications for Therapeutic Intervention.

Psychosis, marked by the emergence of psychotic symptoms, delves into the intricate dance of neurotransmitter dynamics, prominently featuring dopamine as a key orchestrator. In individuals living with psychotic conditions, the finely tuned balance of dopamine becomes disrupted, setting off a cascade of perceptual distortions and the manifestation of psychotic symptoms. A lot of factors can impact dopamine metabolism, further complicating its effects. From genetic predispositions to environmental stressors and inflammation, the delicate equilibrium is susceptible to various influences. The sensorium, the origin of incoming information, loses its intrinsic valence in this complex interplay. The concept of the "signal-to-noise ratio" encapsulates dopamine's role as a molecular switch in neural networks, influencing the flow of information serving the basic biological functions. This nuanced modulation acts as a cognitive prism, shaping how the world is perceived. However, in psychosis, this balance is disrupted, steering individuals away from a shared reality. Understanding dopamine's centrality requires acknowledging its unique status among neurotransmitters. Unlike strictly excitatory or inhibitory counterparts, dopamine's versatility allows it to toggle between roles and act as a cognitive director in the neural orchestra. Disruptions in dopamine synthesis, exchange, and receptor representation set off a chain reaction, impacting the delivery of biologically crucial information. The essence of psychosis is intricately woven into the delicate biochemical ballet choreographed by dopamine. The disruption of this neurotransmitter not only distorts reality but fundamentally reshapes the cognitive and behavioral field of our experience. Recognizing dopamine's role as a cognitive prism provides vital insights into the multifaceted nature of psychotic conditions, offering avenues for targeted therapeutic interventions aimed at restoring this delicate neurotransmitter balance.

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Impact of protein and small molecule interactions on kinase conformations

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

MicroRNA-19a-3p inhibits endothelial dysfunction in atherosclerosis by targeting JCAD

ObjectiveTo examine the influences and mechanisms of MicroRNA-19a-3p (miR-19a-3p) on endothelial dysfunction in atherosclerosis.MethodsAn analysis of miR-19a expression was carried out using the Gene Expression Omnibus (GEO) database. The effect of miR-19a-3p on endothelial function in HUVECs was evaluated by miR-19a-3p overexpression under TNF-α treatment. Luciferase assays were performed to explore the potential target genes. Overexpression of junctional protein associated with coronary artery disease (JCAD) was used to examine the effects of miR-19a-3p on cell adhesion, and proliferation.ResultsMiR-19a-3p expression in endothelial cells decreased after exposure to TNF-α and/or oscillatory flow, consistent with the expression change of miR-19a-3p found in atherosclerotic plaques. Additionally, endothelial cell dysfunction and inflammation were significantly diminished by miR-19a-3p overexpression but markedly exacerbated by miR-19a-3p inhibition. MiR-19a-3p transfection significantly decreased the expression of JCAD by binding to the 3’-UTR of JCAD mRNA. Furthermore, the protective effect of miR-19a-3p against endothelial cell dysfunction and inflammation was achieved by regulating JCAD and was closely linked to the Hippo/YAP signaling pathway.ConclusionMiR-19a-3p expression is a crucial molecular switch in the onset of atherosclerosis and miR-19a-3p overexpression is a possible pharmacological therapeutic strategy for reversing the development of atherosclerosis.

Bis(Dicarbollide) Complexes of Transition Metals: How Substituents in Dicarbollide Ligands Affect the Geometry and Properties of the Complexes.

The interaction between different types of substituents in dicarbollide ligands and their influence on the stabilization of various rotational conformers (rotamers) of transition metal bis(dicarbollide) complexes [3,3'-M(1,2-C2B9H11)2]- are considered. It has been shown that the formation of intramolecular CH···X hydrogen bonds between dicarbollide ligands is determined by the size of the proton acceptor atom X rather than its electronegativity. Due to the stabilization of rotamers with different dipole moments, intramolecular hydrogen bonds between ligands in transition metal bis(dicarbollide) complexes can have a significant impact on the biological properties of their derivatives. In the presence of external complexing metals, weak intramolecular CH···X hydrogen bonds can be broken to form stronger X->M donor-acceptor bonds. This process is accompanied by the mutual rotation of dicarbollide ligands and can be used in sensors and molecular switches based on transition metal bis(dicarbollide) complexes.

The Orange Carotenoid Protein Triggers Cyanobacterial Photoprotection by Quenching Bilins via a Structural Switch of Its Carotenoid.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

Gnao1 is a molecular switch that regulates the Rho signaling pathway in differentiating neurons

GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient’s iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.

Understanding the shape-memory mechanism of thermoplastic polyurethane by investigating the phase-separated morphology: A dissipative particle dynamics study

Shape-memory polyurethanes (SMPUs) are promising materials that change shape in response to external heat. These polymers have a dual-segment structure: a hard segment for netpoint and a soft segment for molecular switch. Understanding the molecular behavior of each segment and microphase-separated morphology is crucial for comprehending the shape-memory mechanism. This study aimed to understand the shape-memory behavior by observing the phase separation of SMPU using mesoscale models based on dissipative particle dynamics (DPD) simulations. The SMPU copolymer was modeled using 4,4′-diphenylmethane diisocyanate (MDI, hard segment) and poly(ethylene oxide) (PEO, soft segment). By calculating segment solubility and repulsion parameters, we found that the hard-segment domain changes from isolated form to a lamellar and interconnected structure and eventually to a continuous form as its content increases. Combining these insights with shape-memory performance models can enhance our understanding of better SMPU design and contribute significantly to the optimization of smart stimuli-responsive materials.

Proton-Induced Reversible Spin-State Switching in Octanuclear FeIII Spin-Crossover Metal-Organic Cages.

Responsive spin-crossover (SCO) metal-organic cages (MOCs) are emerging dynamic platforms with potential for advanced applications in magnetic sensing and molecular switching. Among these, FeIII-based MOCs are particularly noteworthy for their air stability, yet they remain largely unexplored. Herein, we report the synthesis of two novel FeIII MOCs using a bis-bidentate ligand approach, which exhibit SCO activity above room temperature. These represent the first SCO-active FeIII cages and feature an atypical {FeN6}-type coordination sphere, uncommon for FeIII SCO compounds. Our study reveals that these MOCs are sensitive to acid/base variations, enabling reversible magnetic switching in solution. The presence of multiple active proton sites within these SCO-MOCs facilitates multisite, multilevel proton-induced spin-state modulation. This behavior is observed at room temperature through 1H NMR spectroscopy, capturing the subtle proton-induced spin-state transitions triggered by pH changes. Further insights from extended X-ray absorption fine structure (EXAFS) and theoretical analyses indicate that these magnetic alterations primarily result from the protonation and deprotonation processes at the NH active sites on the ligands. These processes induce changes in the secondary coordination sphere, thereby modulating the magnetic properties of the cages. The capability of these FeIII MOCs to integrate magnetic responses with environmental stimuli underscores their potential as finely tunable magnetic sensors and highlights their versatility as molecular switches. This work paves the way for the development of SCO-active materials with tailored properties for applications in sensing and molecular switching.

Potential Transcriptional Enhancers in Coronaviruses: From Infectious Bronchitis Virus to SARS-CoV-2.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions.

Toward High Contrast and Noninvasive Fluorescence Switches via an O-Fused Ring 5,7-Dihydroxy-4-methyl-2,2,3-triphenylbenzofuran-6(2H)-one Strategy.

An unprecedented O-fused ring 5,7-dihydroxy-4-methyl-2,2,3-triphenylbenzofuran-6(2H)-one (3) was first time synthesized. Further, a series of novel dialkyl/fluoroalkyl derivatives of compound 3, 5,7-dialkoxy/fluoroalkoxy-4-methyl-2,2,3-triphenylbenzofuran-6(2H)-one, were obtained with noninvasive fluorescence switching characteristics and aggregation-induced emission properties. Compared with fluoroalkyl derivatives, the alkyl analogs exhibited a significant bathochromic shift in solid-state fluorescence emission. Notably, these noninvasive fluorescent molecular switches could be facilely tuned through light and heat stimulation, which successfully achieved high contrast and reversible fluorescent emission between orange and yellow endowing them with potential applications in data encryption materials. In addition, the single crystal data of compounds 3 and 7-CF3 displayed weak intermolecular interactions in different directions, resulting in twisted conformation and antiparallel slip stacking. Interestingly, the polymer dimethyl silicone film doped with 7-C3F7 also showed an evident light-responsive behavior, meeting the criterion for fluorescent materials in the optical field.

H3K27ac acts as a molecular switch for doxorubicin-induced activation of cardiotoxic genes

BackgroundDoxorubicin (Dox) is an effective chemotherapeutic drug for various cancers, but its clinical application is limited by severe cardiotoxicity. Dox treatment can transcriptionally activate multiple cardiotoxicity-associated genes in cardiomyocytes, the mechanisms underlying this global gene activation remain poorly understood.Methods and resultsHerein, we integrated data from animal models, CUT&Tag and RNA-seq after Dox treatment, and discovered that the level of H3K27ac (a histone modification associated with gene activation) significantly increased in cardiomyocytes following Dox treatment. C646, an inhibitor of histone acetyltransferase, reversed Dox-induced H3K27ac accumulation in cardiomyocytes, which subsequently prevented the increase of Dox-induced DNA damage and apoptosis. Furthermore, C646 alleviated cardiac dysfunction in Dox-treated mice by restoring ejection fraction and reversing fractional shortening percentages. Additionally, Dox treatment increased H3K27ac deposition at the promoters of multiple cardiotoxic genes including Bax, Fas and Bnip3, resulting in their up-regulation. Moreover, the deposition of H3K27ac at cardiotoxicity-related genes exhibited a broad feature across the genome. Based on the deposition of H3K27ac and mRNA expression levels, several potential genes that might contribute to Dox-induced cardiotoxicity were predicted. Finally, the up-regulation of H3K27ac-regulated cardiotoxic genes upon Dox treatment is conservative across species.ConclusionsTaken together, Dox-induced epigenetic modification, specifically H3K27ac, acts as a molecular switch for the activation of robust cardiotoxicity-related genes, leading to cardiomyocyte death and cardiac dysfunction. These findings provide new insights into the relationship between Dox-induced cardiotoxicity and epigenetic regulation, and identify H3K27ac as a potential target for the prevention and treatment of Dox-induced cardiotoxicity.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene-Cyanines Are Loud and Photostable NIR Imaging Agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents, which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches with cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (>3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene–Cyanines Are Loud and Photostable NIR Imaging Agents

AbstractOptoacoustic (or photoacoustic) imaging promises micron‐resolution noninvasive bioimaging with much deeper penetration (&gt;cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR‐absorbing molecular contrast agents, which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches with cyanine dyes to give ultrafast relaxation (&lt;10 ps, &gt;100‐fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (&gt;1000‐fold increase of photostability) and signal loudness (&gt;3‐fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Tuning the Response of Synthetic Mechanogenetic Gene Circuits Using Mutations in TRPV4.

Conventional gene therapy approaches for drug delivery generally rely on constitutive expression of the transgene and thus lack precise control over the timing and magnitude of delivery. Synthetic gene circuits with promoters that are responsive to user-defined stimuli can provide a molecular switch that can be utilized by cells to control drug production. Our laboratory has previously developed a mechanogenetic gene circuit that can deliver biological drugs, such as interleukin-1 receptor antagonist (IL-1Ra), on-demand through the activation of Transient receptor potential family, vanilloid 4 (TRPV4), a mechanosensory ion channel that has been shown to be activated transiently in response to physical stimuli such as physiological mechanical loading or hypo-osmotic stimuli. The goal of this study was to use mutations in TRPV4 to further tune the response of this mechanogenetic gene circuit. Human iPSC-derived chondrocytes harboring targeted gain-of-function mutations of TRPV4 were chondrogenically differentiated. Both mutants-V620I and T89I-showed greater total IL-1Ra production compared with wild type following TRPV4 agonist treatment, as well as mechanical or osmotic loading, but with altered temporal dynamics. Gene circuit output was dependent on the degree of TRPV4 activation secondary to GSK101 concentration or strain magnitude during loading. V620I constructs secreted more IL-1Ra compared with T89I across all experimental conditions, indicating that two mutations that cause similar functional changes to TRPV4 can result in distinct circuit activation profiles that differ from wild-type cells. In summary, we successfully demonstrate proof-of-concept that point mutations in TRPV4 that alter channel function can be used to tune the therapeutic output of mechanogenetic gene circuits.

Differential Lipid Binding Specificities of RAP1A and RAP1B are Encoded by the Amino Acid Sequence of the Membrane Anchors.

RAP1 proteins belong to the RAS family of small GTPases that operate as molecular switches by cycling between GDP-bound inactive and GTP-bound active states. The C-terminal anchors of RAP1 proteins are known to direct membrane localization, but how these anchors organize RAP1 on the plasma membrane (PM) has not been investigated. Using high-resolution imaging, we show that RAP1A and RAP1B form spatially segregated nanoclusters on the inner leaflet of the PM, with further lateral segregation between GDP-bound and GTP-bound proteins. The C-terminal polybasic anchors of RAP1A and RAP1B differ in their amino acid sequences and exhibit different lipid binding specificities, which can be modified by single-point mutations in the respective polybasic domains (PBD). Molecular dynamics simulations reveal that single PBD mutations substantially reduce the interactions of the membrane anchors with the PM lipid phosphatidylserine. In summary, we show that aggregate lipid binding specificity encoded within the C-terminal anchor determines PM association and nanoclustering of RAP1A and RAP1B. Taken together with previous observations on RAC1 and KRAS, the study reveals that the PBD sequences of small GTPase membrane anchors can encode distinct lipid binding specificities that govern PM interactions.

Somatic RAP1B gain-of-function variant underlies isolated thrombocytopenia and immunodeficiency.

The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient's hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.