Biology subjects

Marotta, R.

Publications and source records attributed to Marotta, R..

7 recordsLinked to original sources

bioRxiv2025

Computationally-designed aptamers targeting RAD51-BRCA2 interaction inhibit RAD51 nuclear recruitment

The interaction between RAD51 and BRCA2 plays a key role in homologous recombination (HR), a critical DNA repair mechanism essential for the survival of cancer cells. Disrupting this interaction increases the sensitivity of cancer cells to chemotherapeutic agents. Here, we employed in silico methods to design a novel class of aptamers--customized single-stranded oligonucleotides--specifically engineered to bind RAD51. These aptamers were developed with the aim of selectively modulating RAD51s nuclear recruitment and its role in DNA repair processes. The leading candidate displays high affinity for RAD51, competing with BRCA2 for the same interaction site in vitro, as confirmed through biolayer interferometry (BLI) and fluorescence lifetime imaging microscopy (FLIM). We tested the efficacy of the leading aptamer in pancreatic cancer cells and observed that it significantly impedes RAD51 nuclear localization, reduces homologous recombination (HR) efficiency, and increases DNA damage. Critically, our aptamer potentiates the cytotoxicity of the PARP inhibitor olaparib, exploiting synthetic lethality (SL) to induce cancer cell death. Our study showcases an aptamer-based approach for selectively targeting protein interactions within DNA repair pathways, introducing a promising avenue for SL-based treatments applicable to a wide range of cancers.

biochemistry↗

bioRxiv2023

Novel structural insights on full-length human RAD52: Cryo-EM and beyond

Human RAD52 is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer synthetic lethality strategies since its inhibition or modulation, under specific genetic conditions, was proved to enhance therapies efficacy in various cancer cell types. Although the interest in RAD52 has exponentially grown in the last decade, most information about its structure and mechanism of action is still missing. This work provides novel insights into full-length RAD52 (RAD52 FL) protein, focusing on its structural and functional characterization. The Cryo-Electron Microscopy (Cryo-EM) structure of RAD52 FL, here presented at a resolution (2.16 [A]) higher than the one currently available for RAD52 N-terminal X-ray structure, allows hypothesizing the role of individual amino acid residues. While the N-terminal region of RAD52 FL is structured in an undecameric ring, the C-terminal part is intrinsically disordered as fully characterized through SAXS and biophysical analyses. These detailed (atomic level) structural analyses will substantially impact future characterizations of RAD52 mechanisms of action and inhibitors development, particularly in the context of novel approaches to synthetic lethality.

biophysics↗

bioRxiv2022

Reactive microglia and cytokine release entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the role of the microtubule cytoskeleton during these changes remains unexplored. Here we show that reactive proinflammatory microglia provide a heretofore unique example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, proinflammatory signaling induces recruitment of nucleating material nearby the centrosome and inhibition of centrosomal maturation enhances NLRP3 inflammasome activation and secretion of IL-1{beta}. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation as well as a novel target to modulate cytokine-mediated inflammatory responses in chronic disease and tissue injury.

cell biology↗

bioRxiv2021

Unravelling the regulation pathway of photosynthetic AB-GAPDH

Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B-subunits, and the homo-tetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of B-subunits. Reversible inactivation of AB-GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC-SAXS and single-particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n-GAPDH oligomers with n=1, 2, 4 and 5 co-exist in a dynamic system. B-subunits mediate the contacts between adjacent A2B2 tetramers in A4B4 and A8B8 oligomers. The CTE of each B-subunit penetrates into the active site of a B-subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B-subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A-subunits allows the entrance of the CTE in B-subunits active sites and hence inactive oligomer stabilization. Significance StatementIn land plants, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) unique sink of reducing power of the entire Calvin-Benson cycle, is finely regulated. Based on the redox state and substrates concentration, its heteromeric form AB-GAPDH oscillates between a fully active heterotetramer (A2B2) and inactive oligomers. Experimental evidence demonstrates that GAPDH inactivation depends on the formation of dimers, tetramers or pentamers of A2B2-modules, linked together by C-terminal extensions (CTE) of B-subunits that extrude from one modular tetramer and occupy two active sites of the adjacent one. This molecular mechanism along with the unexpected observed dynamism of the system, shed light on how the Calvin-Benson cycle is modulated in function of the light environmental changes.

biochemistry↗

bioRxiv2021

Astrocytic Regulation of Basal Ganglia Dopamine/D2-Dependent Behaviors

Astrocytic involvement in dopamine dynamics related to motivational and sensorimotor gating abilities is unknown. We found that dysbindin-1 (Dys1) hypofunction increases the activity of astrocytes, which express only the isoform Dys1A that is reduced in the caudate of patients with schizophrenia. Astrocytic Dys1A disruption resulted in avolition and sensorimotor gating deficits, increased astrocytic dopamine D2 receptors and decreased dopaminergic tone within basal ganglia. Notably, astrocytic Dys1A hypofunction disrupted dopamine dynamics linked to reward expectancy and interconnected with astrocytes Ca2+ responses mainly in the globus pallidus externus (GPe). Finally, we proved these phenotypes were mediated by D2 receptors in astrocytes as their selective deletion in astrocytes either in GPe or SNc/VTA enhanced motivation and sensorimotor gating abilities as well as dopaminergic release in the GPe. Therefore, astrocytes control motivational and sensorimotor gating processes through Dys1A/D2-dependent mechanisms within the basal ganglia.

neuroscience↗

bioRxiv2021

RAD51 nuclear recruitment and inhibition towards innovative strategies against pancreatic cancer

RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment. Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a domino effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug. The present study sheds further light on the complexity of the HR pathway, paving the way for designing peptide and small organic molecule inhibitors of RAD51 as innovative anticancer and chemo/radiosensitizer compounds.

biophysics↗

bioRxiv2020

Light-driven ATP production promotes mRNA biosynthesis inside hybrid multi-compartment artificial protocells

The construction of energetically autonomous artificial protocells is one of the most urgent and challenging requirements in bottom-up synthetic biology. Here we show a hybrid multi-compartment approach to build Artificial Simplified-Autotroph Protocells (ASAPs) in an effective manner. Chromatophores obtained from Rhodobacter sphaeroides accomplish the photophosphorylation of ADP to ATP functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles. Under continuous illumination chromatophores produce ATP that in turn sustains the transcription of a DNA gene by T7 RNA polymerase inside ASAPs. Cryo-EM and time-resolved spectroscopy were used for characterizing the chromatophore morphology and the orientation of the photophosphorylation proteins, which allow high ATP production rates (up to ~100 ATP/s per ATP synthase). mRNA biosynthesis inside individual vesicles has been determined by confocal microscopy. The hybrid multi-compartment approach here proposed appears at the same time convenient and effective, and thus very promising for the construction of full-fledged artificial protocells.

synthetic biology↗