Biology subjects

Liccardo, F.

Publications and source records attributed to Liccardo, F..

4 recordsLinked to original sources

bioRxiv2023

Selective activation of intracellular β1AR using a spatially restricted antagonist

G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for {beta}1-adrenergic receptor ({beta}1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable {beta}1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited {beta}1AR on the plasma membrane. In contrast, a cell permeable {beta}1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of {beta}1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while {beta}1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized {beta}1AR signaling in various context, potentially advancing our understanding of {beta}1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

cell biology↗

bioRxiv2023

Inhibition of cAMP signaling prevents congenital heart defects counteracting oxidative stress in Pde2A deficient embryos

BackgroundPhosphodiesterases (PDEs) are the enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) playing a key role in the homeostasis of these two second messengers. PDE2A is a dual-specific PDE that breaks down both cAMP and cGMP and can be activated by cGMP. It appears peculiar that the Pde2A-deficient (Pde2A-/-) mouse model is embryonically lethal, likely due to a strongly reduced size of liver and to a severe anemia. In addition, the heart of Pde2A-/- embryos shows ventricular and atrial septum defects, hypertrabeculation, heart dilatation and non-compaction defects. We recently highlighted a direct relationship between Pde2A impairment, consequent increase of cAMP and the onset of mouse congenital heart defects (CHDs), however the molecular mechanisms underlining the heart defects remain unknown. MethodsTranscriptome analysis of Pde2A-/- embryonic heart was performed by RNA sequencing and the most altered genes were also analyzed by quantitative real time PCR. In vivo treatment with drugs acting on cAMP signaling (Metoprolol and H89) and oxidative stress (N-Acetyl-Cysteine, NAC) were carried out on pregnant Pde2A+/- female. Histological, biochemical, and molecular analyses were then performed on embryonic hearts. ResultsWe found a significant modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 administration were able to prevent heart dilatation and hypertabeculation in Pde2A-/-embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Partial rescue of cardiac defects was observed by using the antioxidant NAC, indicating oxidative stress like one of the molecular mechanisms underpinning the CHDs. ConclusionsWe identified specific biological processes, molecules and cell signaling that can be targeted by selected drugs with consequent beneficial effects for cAMP-dependent CHDs. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LICongenital Heart Defects are the most frequent heart birth defects including septal defects, hypertrabeculation and non-compacted myocardium. C_LIO_LIPde2A hydrolyses the cAMP and cGMP second messengers. C_LIO_LIPde2A-deficient mice are embryonic lethal and show cAMP-dependent Congenital Heart Defects. C_LI What New Information Does This Article Contribute?O_LIWe identified several novel pathways altered in hearts of Pde2A-/- embryos. C_LIO_LIWe demonstrated that drugs lowering cAMP levels rescued specific CHDs in Pde2A-/- embryos. C_LIO_LIWe discovered that antioxidants are beneficial for CHDs in Pde2A-/- embryos. C_LI The significance of this work relay in molecular discoveries and pharmacological approaches to treat CHDs by using a mouse model that recapitulate the major congenital heart defects. Among the pathways involved in specific defects associated with CHDs, the transcriptome analysis revealed an impairment of genes of the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in Pde2A-/- hearts. The scientific community will have open access to the RNA-seq data that can be utilized to further understand the congenital cardiac pathology and clarify the molecular implication in selected defects such as septal and ventricular wall defects. Up to date CHDs, when possible and if identified in time, are mostly treated trough surgery. The identification of drugs blunting the cAMP signaling response or reducing oxidative stress pathways will be useful for setting therapeutic approaches to alleviate CHDs.

developmental biology↗

bioRxiv2023

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection

Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). It exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with bone marrow stromal cells (BMSC). Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML cells and BMSC upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSC. Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML cells and BMSC, involving mechanotransduction and YAP signaling

cancer biology↗

bioRxiv2021

Precision localization of cellular proteins with fluorescent Fab-based probes

Currently, a major technical limitation of microscopy based image analysis is linkage error - a visualization error that is measured by the distance between the cellular protein to be detected and the fluorescence emitter used for detection. With continuously improving resolution of todays (super-resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five-fold when compared to conventional staining techniques. These reagents, called Fluo-N-Fabs, consist of an antigen binding fragment of a full-length antibody (Fab/ fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo-N-Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.

cell biology↗