Biology subjects

Sironi, M.

Publications and source records attributed to Sironi, M..

11 recordsLinked to original sources

bioRxiv2026

Ligand Binding Free Energy Landscapes at the Tubulin Colchicine Site from Coarse-Grained Metadynamics

Accessing deeply buried binding sites remains a major challenge in structure-based drug discovery, where accurate description of both protein dynamics and ligand binding pathways is required. Funnel metadynamics enables simulation of complete binding processes but is computationally demanding at the all-atom resolution. By adopting the Martini 3 force field, coarse-grained funnel metadynamics (CG-FMD) substantially reduces computational requirements while retaining enhanced sampling capabilities. In this work, we assess the capability of CG-FMD to model ligand recognition at the deeply buried colchicinoids site of the tubulin {beta}-heterodimer, a multisite protein of strategic importance. We investigated the binding of colchicine, podophyllotoxin and combretastatin-A4, recovering free energy profiles with improved statistical convergence compared to AA-FMD and comparable to experimental references. In particular CG-FMD binding free energies present mean absolute errors between 3 and 10 kJ mol-1. These results propose CG-FMD as an efficient, physics-based framework for probing ligand binding to challenging sites.

biophysics↗

bioRxiv2025

Reassessing taxonomy and virulence in the Fusobacterium nucleatum group - Rebuttal of Fusobacterium animalis clades "Fna C1" and "Fna C2", genome announcement for Fusobacterium watanabei and description of Fusobacterium paranimalis sp. nov.

There is a considerable interest in the association between Fusobacterium animalis and colorectal cancer (CRC). Recently, it was suggested that this association is valid only for a distinct clade of F. animalis (Fna C2) and that F. animalis strains belonging to another clade (Fna C1) are only associated with the oral cavity. It was further suggested that this made Fna C1 a natural comparator when looking for candidate genes associated with the pathogenicity of Fna C2. Based on such comparisons, three candidate operons enriched in CRC were suggested to explain the strong colorectal tumor association of F. animalis. In the present paper we show that major taxonomic errors invalidate the existence of two distinct clades of F. animalis and that Fna C1 is simply a rediscovery and misclassification of Fusobacterium watanabei. We further reassess the phylogenetic structure of the entire Fusobacterium nucleatum group encompassing F. animalis and all known closely related species and confirm the current taxonomy using contemporary phylogenetic principles. We also describe a novel Fusobacterium species more closely related to F. animalis than any other known species, for which we propose the name Fusobacterium paranimalis sp. nov.. We further searched for the three proposed candidate virulence operons of F. animalis across the entire F. nucleatum group and show that some or all of these are present in all other species except F. watanabei. We also observe considerable variability of Type 5 secretion systems (T5SS) by subtype and abundance across the F. nucleatum group. ImportanceIt is known that Fusobacterium animalis is able to survive within colorectal tumors. Recently, it was proposed that only one "clade" of Fusobacterium animalis could be found in colorectal tumors, and that another "clade" within the same species was instead only found in the oral cavity, and that differences in gene content could explain the habitat difference. We here show that these "clades" are two separate species by sequencing the type strain of the oral cavity-associated species, which is Fusobacterium watanabei. We also revisit other related species within the "Fusobacterium nucleatum group" to confirm that they are separate species, exemplified by presenting a new species, F. paranimalis sp.nov., which genetically is more related to F. animalis than any other known species including F. watanabei. We look at gene content of the entire group, and conclude that known virulence genes cannot fully explain F. animalis cancer association.

genomics↗

bioRxiv2024

Tangled genetic relationships within the Fusobacterium genus

Fusobacteria have been associated to different diseases, including colorectal cancer (CRC), but knowledge of which taxonomic groups contribute to specific conditions is incomplete. We analyzed the genetic diversity and relationships within the Fusobacterium genus. We report recent and ancestral recombination in core genes, indicating that fusobacteria have mosaic genomes and emphasizing that taxonomic demarcation should not rely on single genes/gene regions. Across databases, we found ample evidence of species miss-classification and of undescribed species, which are both expected to complicate disease association. By focusing on a lineage that includes F. periodonticum/pseudoperiodonticum and F. nucleatum, we show that genomes belong to four modern populations, but most known species/subspecies emerged from individual ancestral populations. Of these, the F. periodonticum/pseudoperiodonticum population experienced the lowest drift and displays the highest genetic diversity, in line with the less specialized distribution of these bacteria in oral sites. A highly drifted ancestral population instead contributed genetic ancestry to a new species, which includes genomes classified within the F. nucleatum animalis diversity in a recent CRC study. Thus, evidence herein calls for a re-analysis of F. nucleatum animalis features associated to CRC. More generally, our data inform future molecular profiling approaches to investigate the epidemiology of Fusobacterium-associated diseases.

microbiology↗

bioRxiv2023

RORγ bridges cancer-driven lipid dysmetabolism and myeloid immunosuppression

Despite well-documented metabolic and hematopoietic alterations during tumor development1, the mechanisms underlying this crucial immunometabolic intersection have remained elusive. Of particular interest is the ligand-activated transcription factor retinoic acid-related orphan receptor 1 (RORC1/ROR{gamma}), whose activity is boosted by cholesterol metabolites2, acting as a modulator of cancer-related emergency myelopoiesis3, while hypercholesterolemia itself is associated with dysregulated myelopoiesis4,5. Here we show that both cancer growth and hypercholesterolemic diet can independently or cooperatively activate ROR{gamma}-dependent expansion of myeloid-derived suppressor cells (MDSCs) and M2 polarization of tumor-associated macrophages (TAMs), thereby supporting cancer spread. Moreover, we report that tumor development enhances the hepatic production of IL-1{beta} and IL-6, which in turn promote upregulation of hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, as we confirmed in models of fibrosarcoma, melanoma, colorectal (CRC), and lung cancer, as well as in CRC, non-small-cell lung cancer (NSCLC), breast (BRC), pancreatic ductal adenocarcinoma (PDAC), biliary tract carcinoma (BTC) and pancreatic neuroendocrine tumor (PNET) patients. Importantly, lowering cholesterol levels prevents MDSC expansion and M2 TAM accumulation in a ROR{gamma}-dependent manner, unleashing specific anti-tumor immunity and improving the efficacy of anti-PD-1 immunotherapy. Overall, we identify ROR{gamma} as a novel sensor of lipid disorders in cancer bearers, bridging hypercholesterolemia and pro-tumor myelopoiesis.

immunology↗

bioRxiv2022

Genomic clustering and sequence context of mutations in human monkeypox virus (hMPXV1) genomes

The ongoing worldwide monkeypox outbreak is caused by viral lineages (globally referred to as hMPXV1) that are related to but distinct from clade IIb MPXV viruses transmitted in Nigeria. Analysis of genetic differences indicated that APOBEC-mediated editing might be responsible for the unexpectedly high number of mutations observed in hMPXV1 genomes. Here, using 1624 hMPXV1 publicly available sequences, we analyzed mutations that accrued since 2017 until the emergence of the current predominant variant (B.1), as well as those that that have been accumulating during the 2022 outbreak. We found that substitutions tend to cluster and mutational hot-spots are observed. Investigation of the sequence context of C to T changes indicated a preference for 5-TCA/G-3 motifs, suggesting APOBEC3F- or APOBEC3A-mediated editing. The sequence context has remained unchanged since 2017, indicating that the same mutational mechanism that is driving the accumulation of substitutions during the ongoing human-to-human transmission, was already operating before the virus left Africa. We suggest that APOBEC3A is the most likely candidate, given its expression in the skin and its known role in the editing of human papillomavirus.

genomics↗

bioRxiv2022

SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress and autophagic flow

Coronaviruses encode a variable number of accessory proteins that play a role in host-virus interactions, in the suppression of immune responses, or in immune evasion. Accessory proteins in SARS-CoV-2 consist of at least twelve viral proteins whose roles during infection have been extensively studied. Nevertheless, the role of the ORF3c accessory protein, an alternative open reading frame of ORF3a, has remained elusive. Herein, we characterized ORF3c in terms of cellular localization, hosts antiviral response modulation, and effects on mitochondrial metabolism. We show that ORF3c has a mitochondrial localization and alters mitochondrial metabolism, resulting in increased ROS production, block of the autophagic flux, and accumulation of autophagosomes/autolysosomes. Notably, we also found that ORF3c induces a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. This is similar to the condition observed in the chronic degenerative phase of COVID-19. Altogether these data suggest that ORF3c could be a key protein for SARS-CoV-2 pathogenesis and that it may play a role in disease progression.

microbiology↗

bioRxiv2022

Variola virus is as old as the earliest historical lines of evidence suggest

Archaeovirology efforts provided a rich portrait of the evolutionary history of variola virus (VARV), the causative agent of smallpox. These studies revealed frequent viral lineage extinction and a relatively recent origin of VARV as a human pathogen ([~] 1,700 years ago, ya). This contrasts with historical records suggesting the presence of smallpox as early as 3,500 ya. By performing an analysis of ancestry components in modern, historic, and ancient VARV genomes, we unveil the progressive drifting of VARV lineages from a common ancestral population and we show that a small proportion of Viking-Age ancestry persisted until the 18th century. After the split of the P-I and P-II lineages, which occurred before the onset of smallpox vaccination, the former (but not the latter) experienced a severe bottleneck. We suggest this was due to the distinct epidemiology of the two lineages, which display remarkably different disease severity. As for the emergence of VARV, we corrected time estimates by accounting for the time-dependent rate phenomenon. Using this approach, we estimate that VARV emerged earlier than 3,800 ya, thus supporting its presence in ancient societies, including Egypt, as pockmarked mummies suggest. ImportanceNow eradicated, smallpox was one of the most devastating human diseases, causing the death of at least 300 million people in the 20th century. Humans were the only known host of variola virus, but the time-frame of its emergence in our species has been a matter of debate. Specifically, molecular dating suggested a relatively recent origin, whereas historical sources indicated the presence of VARV in ancient societies. By applying population genetic methods, we analyzed the ancestry components in modern and historic VARV genomes, and we found a progressive drifting of VARV lineages from a common ancestral population. By accounting for the common observation that rates of viral evolution scale negatively with the time-frame of measurement, we estimated that VARV emerged earlier than 3800 years ago. Thus, out data settle a controversy and provide novel insight into the origin and evolution of one of the most historically relevant human pathogens.

evolutionary biology↗

bioRxiv2022

Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3

Streptococcus pneumoniae is a major pathogen in children, elderly subjects and immunodeficient patients. PTX3 is a fluid phase pattern recognition molecule (PRM) involved in resistance to selected microbial agents and in regulation of inflammation. The present study was designed to assess the role of PTX3 in invasive pneumococcal infection. In a murine model of invasive pneumococcal infection, PTX3 was strongly induced in non-hematopoietic (particularly, endothelial) cells. The IL-1{beta}/MyD88 axis played a major role in regulation of the Ptx3 gene expression. Ptx3-/- mice were more susceptible to invasive pneumococcal infection. Although high concentrations of PTX3 had opsonic activity in vitro, no evidence of PTX3-enhanced phagocytosis was obtained in vivo. In contrast, Ptx3-deficient mice showed enhanced recruitment of neutrophils and inflammation. Using P-selectin deficient mice, we found that protection against pneumococcus was dependent upon PTX3-mediated regulation of neutrophil inflammation. In humans, PTX3 genetic polymorphisms were associated with invasive pneumococcal infections. Thus, this fluid phase PRM plays an important role in tuning inflammation and resistance against invasive pneumococcal infection.

immunology↗

bioRxiv2020

Simplexviruses successfully adapt to their host by fine-tuning immune responses

Primate herpes simplex viruses are relatively harmless to their natural hosts, whereas cross-species transmission can result in severe disease. We performed a genome-wide scan for signals of adaptation of simplexviruses to hominins. We found evidence of positive selection in three glycoproteins, with selected sites located in antigenic determinants. Positively selected non-core proteins were involved in different immune-escape mechanisms. By expressing mutants of one of these proteins (ICP47), we show that the amino acid status at the positively selected sites is sufficient to induce HLA-G. HSV-1/HSV-2 ICP47 induced HLA-G when mutated to recapitulate residues in B virus, whereas the mutated version of B virus ICP47 failed to determine HLA-G expression. Thus, the evolution of ICP47 in HSV-1/HSV-2 determined the loss of an immunosuppressive effect, suggesting that simplexviruses tune immune responses to promote successful co-existence with their hosts. These results also help explain the high pathogenicity of B virus in humans.

microbiology↗

bioRxiv2020

Antigenic variation of SARS-CoV-2 in response to immune pressure

The ongoing evolution of SARS-CoV-2 is expected to be at least partially driven by the selective pressure imposed by the human immune system. We exploited the availability of a large number of high-quality SARS-CoV-2 genomes, as well as of validated epitope predictions, to show that B cell epitopes in the spike glycoprotein (S) and in the nucleocapsid protein (N) have higher diversity than non-epitope positions. Similar results were obtained for other human coronaviruses. Conversely, in the SARS-CoV-2 population, epitopes for CD4+ and CD8+ T cells were not more variable than non-epitope positions. A significant reduction in epitope variability was instead observed for some of the most immunogenic proteins (S, N, ORF8, and ORF3a). Analysis over longer evolutionary time-frames indicated that this effect is not due to differential constraints. These data indicate that SARS-CoV-2 is evolving to elude the host humoral immune response, whereas recognition by T cells might benefit the virus.

microbiology↗

bioRxiv2020

Substrate Recognition by γ-Glutamyltransferase: A Molecular Dynamics Simulations Study

{gamma}-glutamyltransferase (GGT) is an enzyme that uses {gamma}-glutamyl compounds as substrate and catalyzes their transfer into a water molecule or an acceptor substrate with varied physiological-function in bacteria, plants and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, structural dynamics of the substrate binding to the catalytic site of GGT is unknown. Here, we modeled Escherichia Coli GGTs glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggests its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

biophysics↗