Biology subjects

Paiardini, A.

Publications and source records attributed to Paiardini, A..

9 recordsLinked to original sources

bioRxiv2026

m6A RNA methylation modulates Zika virus infection by regulating serine proteases in Aedes albopictus

Epitranscriptomic RNA modifications, particularly N6-methyladenosine (mA), have emerged as important regulators of host-virus interctions. However, the role of mA in arbovirus infection within mosquito vectors remains poorly defined. Here, we characterized the mA RNA methylation machinery in Aedes albopictus C6/36 cells and examined its contribution to Zika virus (ZIKV) replication. Arbovirus infection did not significantly alter the transcriptional levels or enzymatic activity of the core mA methyltransferase components METTL3 and METTL14. In contrast, pharmacological inhibition of METTL3 markedly enhanced ZIKV replication, indicating an antiviral role for mA in mosquito cells. Transcriptome-wide analysis of C6/36 cells treated with the METTL3 inhibitor STM2457 revealed extensive changes in gene expression, including the pronounced upregulation of multiple serine proteases, particularly members of the CLIP family. Single-nucleotide-resolution mapping of mA using GLORI-sequencing showed that m6A is absent from Zika virus RNA, but readily detectable in the A. albopictus transcriptome. Data analysis defined key features of the mosquito epitranscriptome and demonstrated that mA modifications are enriched within the coding regions of serine protease transcripts, supporting their direct regulation by mA. Functionally, inhibition of serine protease activity using AEBSF resulted in a significant reduction of ZIKV replication. Together, these findings identify mA RNA methylation as a critical regulator of ZIKV infection in mosquito cells and uncover an epitranscriptomic pathway linking mA-dependent control of serine proteases to vector-virus interactions. Author SummaryMosquito-borne viruses such as Zika virus pose a major threat to global public health. Successful transmission of these viruses depends not only on infection in humans, but also on their ability to replicate efficiently inside mosquito vectors. Chemical modifications of RNA, collectively known as epitranscriptomic marks, have recently emerged as important regulators of gene expression and virus-host interactions. Among these, N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic cells. While m6A has been extensively studied in mammalian systems, its role in mosquito antiviral responses remains poorly understood. In this study, we investigated how m6A RNA methylation influences Zika virus infection in mosquito cells derived from Aedes albopictus. We found that reducing m6A levels enhances viral replication, indicating that this RNA modification restricts infection in mosquito cells. Notably, Zika virus RNA itself does not contain detectable m6A modifications. Instead, m6A regulates the expression of specific mosquito genes, including a group of serine proteases that influence viral replication. Pharmacological inhibition of these proteases significantly impaired virus growth, identifying them as key downstream effectors. Our findings reveal an antiviral role for m6A in mosquito cells and uncover a previously unrecognized epitranscriptomic pathway that shapes mosquito-virus interactions. Understanding how RNA modifications regulate arbovirus infection in vectors may open new avenues for strategies aimed at limiting virus transmission.

molecular biology↗

bioRxiv2025

Unravelling the role of epigenetic regulators during embryonic development of Rhipicephalus micropolus

Epigenetic modifications are long-lasting changes to the genome that influence a cells transcriptional potential, thereby altering its function. These modifications can trigger adaptive responses that impact protein expression and various cellular processes, including differentiation and growth. The primary epigenetic mechanisms identified to date include DNA and RNA methylation, histone modifications, and microRNA-mediated regulation of gene expression. The intricate crosstalk among these mechanisms makes epigenetics a compelling field for the development of novel control strategies, particularly through the use of epigenetic drugs targeting arthropod vectors such as ticks. In this study, we identified the Rhipicephalus microplus orthologs of canonical histone-modifying enzymes, along with components of the machinery responsible for m5C and 6mA-DNA, and m6A-RNA methylations. We further characterized their transcriptional profiles and enzymatic activities during embryonic development. To explore the functional consequences of epigenetic regulation in R. microplus, we evaluated the effects of various epigenetic inhibitors on the BME26 tick embryonic cell line. Molecular docking simulations were performed to predict the binding mode of these inhibitors to tick enzymes, followed by in vitro assessment of their effects on cell viability and morphology. Tick cells exposed to these inhibitors exhibited phenotypic and molecular alterations. Notably, we observed higher levels of DNA methylation in the mitochondrial genome compared to nuclear DNA. Inhibition of DNA methylation using 5-azacytidine (5-AZA) was associated with increased activity of the mitochondrial electron transport chain and ATP synthesis, but reduced cellular proliferation. Our findings highlight the importance of epigenetic regulation during tick embryogenesis and suggest that targeting these pathways may offer a novel and promising strategy for tick control. HighlightsO_LIR. microplus presents a complete set of epigenetic enzymes that modify histones, DNA and RNA C_LIO_LIEpigenetic regulation is highly dynamic, and functionally significant during R. microplus embryogenesis C_LIO_LIInhibition of DNA methylation leads to overactivation of the mitochondrial electron transport chain C_LI

molecular biology↗

bioRxiv2025

An evolutionarily conserved role for separase in the regulation of nuclear lamins

Separase is a well conserved endopeptidase that facilitates sister chromatid separation at the metaphase-anaphase transition by cleaving cohesins. Beyond its role in chromosome segregation, Separase also participates in various biological processes, including chromatin organization and replication, centrosome disengagement and duplication, cytokinesis, and telomere capping. Here, we report that the loss of Drosophila separase (Sse) function induces significant changes in global protein expression and affects the protein levels of both A/C-type lamin C (LamC) and B-type lamin Dm0 (Dm0). We further demonstrate that SSE physically interacts with lamins and colocalizes with them at the nuclear envelope during interphase. Additionally, loss of SSE activity disrupts nuclear organization in larval muscles and impairs locomotion in adult flies. Notably, similar to SSE in flies, depletion of human separase (ESPL1) in SV40 fibroblasts leads to misshapen nuclei and increased levels of lamin A. Moreover, we show that ESPL1 interacts with lamin A in human fibroblasts, suggesting that the functional interaction between Separase and lamins is evolutionarily conserved across different organisms.

genetics↗

bioRxiv2023

THE RIBOREGULATION MECHANISM OF HUMAN SERINE HYDROXYMETHYLTRANSFERASE IS ROOTED IN AN ALLOSTERIC SWITCH

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzymes reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

biochemistry↗

bioRxiv2022

A short ERAP2 that binds IRAP is expressed in macrophages independently from gene variation

The M1 zinc metalloproteases ERAP1, ERAP2 and IRAP play a role in HLA-I antigen presentation by refining the peptidome either in the ER (ERAP1 and ERAP2) or in the endosomes (IRAP). They have been also entrusted with other, although less defined, functions such as the regulation of the angiotensin system and blood pressure. In humans, ERAP1 and IRAP are commonly expressed. ERAP2 instead has evolved under balancing selection that maintains two haplotypes one of which undergoing RNA splicing leading to nonsense-mediated decay and loss of protein. Hence, likewise in rodents in which the ERAP2 gene is missing, about a quarter of the human population does not express ERAP2. We report here that macrophages, but not monocytes or other mononuclear blood cells, express and secrete an ERAP2 shorter form independently from the haplotype. The generation of this "short" ERAP2 is due to an autocatalytic cleavage within a distinctive structural motif and requires an acidic microenvironment. Remarkably, ERAP2 "short" binds IRAP and the two molecules are co-expressed in the endosomes as well as in the cell membrane. Of note, the same phenomenon could be observed in some cancer cells. These data prompt to reconsider the role of ERAP2 which might have been maintained in humans because fulfilling a relevant function as "short" form in specialized cells.

biochemistry↗

bioRxiv2022

Revisiting degron motifs in human AURKA required for its targeting by APC/C-FZR1

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and association with cancer, and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the Anaphase-Promoting Complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the A-box in addition to a reported canonical D-box degron in the C-terminus. Here we find that the proposed C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, as previously reported in vitro, the N-terminal intrinsically disordered region (IDR) of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL Short Linear Interacting Motif (SLiM) of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA additionally depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA. Summary blurbAURKA degron motifs are redefined to show that the so-called N-terminal A-box is in fact a D-box, and the so-called D-box in the C-terminus is not a degron but a motif critical for the active, degradable conformation of AURKA

cell biology↗

bioRxiv2021

A Focused Small-Molecule Screen Identifies PHA-680626 as an Amphosteric Inhibitor Disrupting the Interaction between Aurora-A and N-Myc

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcriptional factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation, by directly binding to a highly conserved N-Myc region, i.e. Myc Box I. As a result, elevated levels of N-Myc, which are required for the growth of MYCN amplified cells, are observed. During the last years, it has been demonstrated that the ATP competitive inhibitors of AURKA CD532, MLN8054 and Alisertib also cause essential conformational changes in the structure of the activation loop of the kinase that prevent N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complex with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results, identifying PHA-680626 as an amphosteric inhibitor both in vitro and MYCN overexpressing cell lines, expand the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex, and confirm that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

cancer biology↗

bioRxiv2020

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), the latter two targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result strongly indicates that the role of the dTMP-SC assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, may provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

biochemistry↗

bioRxiv2019

Revisiting the \"satisfaction of spatial restraints\" approach of MODELLER for protein homology modeling

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the \"modeling by satisfaction of spatial restraints\" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of{sigma} values profoundly influences 3D modeling. We show that the{sigma} values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the programs predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLERs objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance.\n\nAuthor summaryProteins are fundamental biological molecules that carry out countless activities in living beings. Since the function of proteins is dictated by their three-dimensional atomic structures, acquiring structural details of proteins provides deep insights into their function. Currently, the most successful computational approach for protein structure prediction is template-based modeling. In this approach, a target protein is modeled using the experimentally-derived structural information of a template protein assumed to have a similar structure to the target. MODELLER is the most frequently used program for template-based 3D model building. Despite its success, its predictions are not always accurate enough to be useful in Biomedical Research. Here, we show that it is possible to greatly increase the performance of MODELLER by modifying two aspects of its algorithm. First, we demonstrate that providing the program with accurate estimations of local target-template structural divergence greatly increases the quality of its predictions. Additionally, we show that modifying MODELLERs scoring function with statistical potential energetic terms also helps to improve modeling quality. This work will be useful in future research, since it reports practical strategies to improve the performance of this core tool in Structural Bioinformatics.

bioinformatics↗