Biology subjects

Forneris, F.

Publications and source records attributed to Forneris, F..

10 recordsLinked to original sources

bioRxiv2026

Extracellular chaperones modulate AL light chains fibrillar aggregation and contribute to amyloid structural heterogeneity

In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LCs intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo, likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

biochemistry↗

bioRxiv2026

Structural studies of the C-terminal cysteine-rich domain of Aedes albopictus vitellogenin reveal an evolutionarily divergent, monomeric C-terminal cysteine knot (CTCK) architecture

Vitellogenins are essential transport lipoproteins and precursors to egg-yolk proteins in oviparous species. Several molecular structure studies have elucidated most of their multi-domain organization, yet the structure and function of their C-terminal cysteine knot (CTCK) domain have remained largely elusive. In this study, we present the 1.2 [A] resolution crystal structure of the recombinant CTCK domain from a vitellogenin isoform of the mosquito Aedes albopictus (Vg-CTCK). The molecular architecture reveals a CTCK fold defined by two antiparallel beta sheets stabilized by three intramolecular disulfide bonds, featuring a unique 12-amino acid insertion shaping an alpha helix positioned between the two main beta sheets. Analysis of the crystal packing and biophysical characterization in solution consistently confirms that recombinant Vg-CTCK is monomeric. To validate these findings in a native context, we employed ex vivo cross-linking mass spectrometry (XL-MS) on intact mosquito ovaries, which corroborated the molecular architecture of the Vg-CTCK observed in the crystal structure and highlighted the absence of inter-molecular cross-links. Collectively, our data highlight an evolutionarily divergent, monomeric assembly for the mosquito Vg-CTCK domain, challenging previous hypotheses that suggested this domain might facilitate vitellogenin oligomerization.

molecular biology↗

bioRxiv2026

Cardiac Calsequestrin is a Physiological Dimer that Polymerizes through a Ca2+-Triggered Cooperative Switch

Cardiac Calsequestrin (CASQ2) polymerizes within the junctional sarcoplasmic reticulum to buffer Ca{superscript 2} and regulate ryanodine receptor 2 (RyR2) gating, yet the molecular mechanism governing this process remains poorly understood. Using an integrated set of complementary approaches spanning single-particle biophysics, bulk solution measurements, and polymer chemistry, we demonstrate that CASQ2 is an intrinsic dimer at nanomolar concentrations and under physiological ionic conditions, independently of Ca{superscript 2}. In addition, Ca{superscript 2}-dependent polymerization operates as a highly cooperative switch between a stable oligomeric phase and a high-order polymeric state. Physiological amounts of K ions modulate this switch through a biphasic electrostatic mechanism, supporting polymerization at low concentrations and inhibiting it beyond charge neutralization ([~]194 mM). These findings redefine CASQ2 as an intrinsic dimer with polymerization-switch properties, and provide a mechanistic framework for understanding how catecholaminergic polymorphic ventricular tachycardia type 2 mutations, distributed evenly across the CASQ2 surface, cause disease through two distinct pathological trajectories.

biochemistry↗

bioRxiv2025

Spiroplasma impairs testes gene expression in Glossina fuscipes fuscipes

Glossina fuscipes fuscipes is a riverine tsetse fly species, the primary vector of human and animal trypanosomiasis in Sub-Saharan Africa. Controlling tsetse fly populations is crucial for mitigating the socio-economic impact of this disease, as effective treatments remain challenging. Indeed, the development of control strategies is hindered by the species unique reproductive biology: adenotrophic viviparity, in which the female retains and nourishes the developing larva in her uterus throughout the pregnancy. The discovery of Spiroplasma in some G. f. fuscipes populations has drawn attention as a potential tool to enhance tsetse fly control strategies. Although Spiroplasma does not exhibit in G. f. fuscipes the male-killing phenotype observed in Drosophila melanogaster, evidence suggests that it may confer refractoriness to Trypanosoma infection. This has led to further investigations into its broader effects on G. f. fuscipes biology, particularly its potential impact on Glossina reproductive fitness. In this study, we considered Spiroplasma effect on the male reproduction. For this, we performed a differential gene expression analysis on testes and male accessory glands (MAGs) between Spiroplasma-infected and uninfected males. A significant downregulation of genes was observed in testes while a minor effect has been detected on MAGs. Downregulation of testes genes associated with functions related to sperm motility, energy metabolism, and mitochondrial function has been observed. Additionally, differentially expressed genes involved in antimicrobial activity and circadian rhythm regulation were observed. These findings provide valuable insights into the potential fitness costs of Spiroplasma infection for the fly and its implications for the bacterium use as biological control strategies targeting G. f. fuscipes.

genomics↗

bioRxiv2024

Molecular Structure and Enzymatic Mechanism of the Human Collagen Hydroxylysine Galactosyltransferase GLT25D1/COLGALT1

During biosynthesis, collagen lysine residues undergo extensive post-translational modifications essential for the stability and functions of collagen supramolecular assemblies. In the endoplasmic reticulum, two distinct metal ion dependent enzyme families (i.e., multifunctional lysyl hydroxylases-glucosyltransferases LH/PLODs and galactosyltransferases GLT25D/COLGALT) alternatively operate on collagen lysine side chains ultimately generating the highly conserved -(1,2)-glucosyl-{beta}-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in the collagen lysine post-translational modification machinery is linked to multiple developmental pathologies as well as extracellular matrix alterations causing enhanced cellular proliferation and invasiveness of several solid tumors, prompting for an in-depth characterization of LH/PLOD and GLT25D/COLGALT enzyme families. Here, we present an integrative molecular study of GLT25D1/COLGALT, highlighting an elongated head-to-head homodimeric assembly characterized by an N-terminal segment of each monomer wrapping around its dimerization partner. Each monomer encompasses two Rossman fold-type domains (GT1 and GT2) separated by an extended linker. Both domains were found capable of binding Mn2+ cofactors and UDP--galactose donor substrates, resulting in four candidate catalytic sites per dimer. Site-directed mutagenesis and biochemical studies identify the C-terminal GT2 domain as the functional GLT25D1/COLGALT1 catalytic site, highlighting an unprecedented Glu-Asp-Asp motif critical for metal ion binding, and suggesting structural roles for the N-terminal GT1 essential for correct quaternary structure assembly. Conversely, dimerization was not a requirement for GLT25D1/COLGALT1 enzymatic activity in vitro, suggesting that the elongated enzyme homodimer assembly, resembling that of LH/PLOD binding partners, could represent a functional hallmark for correct recognition and successful processing of collagen lysine residues.

biochemistry↗

bioRxiv2021

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

microbiology↗

bioRxiv2020

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine-protease best known for its selective processing of the potent synaptic organiser agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. Something which, based on the available literature, likely stems from NT-based regulation of agrin signalling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, owing to the scarce molecular detail regarding NT activity and NT-agrin interactions. The difficult recombinant production of NT in its catalytically competent form is at the base of these limitations, and is currently constraining a more detailed molecular, biochemical and structural characterisation of the NT-agrin system. We have developed a novel strategy to reliably produce and purify a truncated but catalytically competent variant of NT called NT-mini. The characterisation of our construct highlighted almost wild type-like behaviour with comparable specificity, and conservation of modulation by calcium and heparin despite the lack of several accessory domains. With the data obtained from NT-mini it was then possible to identify NTs heparin-binding domain, and discover a novel putative Zinc-based modulation of NT. Additionally, NT-mini allowed us to investigate the effect of NT activity on myotube formation in controlled cell-based experiments, evidencing a negative impact on myoblast fusion dependant on enzymatic activity. Collectively, this shows the viability of NT-mini as a model to study NT functionality, allowing to expand both in-vitro and "in-cellulo" investigations and providing a foundation to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

biochemistry↗

bioRxiv2020

A minimal hepatocyte growth factor mimic acting as a powerful agonist of the MET receptor tyrosine kinase for regeneration of epithelial tissues and organs.

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent on MET activation.

bioengineering↗

bioRxiv2019

A cooperative network of molecular "hot spots" highlights the complexity of LH3 collagen glycosyltransferase activities

Hydroxylysine glycosylations are collagen-specific post-translational modifications essential for maturation and homeostasis of fibrillar as well as non-fibrillar collagen molecules. Lysyl hydroxylase 3 (LH3) is the only human enzyme capable of performing two chemically-distinct collagen glycosyltransferase reactions using the same catalytic site: inverting beta-1,O-galactosylation of hydroxylysines and retaining alpha-1,2-glycosylation of galactosyl hydroxylysines. Here, we used structure-based mutagenesis to show that both glycosyltransferase activities are strongly dependent on a broad cooperative network of amino acid side chains which includes the first-shell environment of the glycosyltransferase catalytic site and shares features with both retaining and inverting enzymes. We identified critical "hot spots" leading to selective loss of inverting activity without affecting the retaining reaction. Finally, we present molecular structures of LH3 in complex with UDP-sugar analogs which provide the first structural templates for LH3 glycosyltransferase inhibitor development. Collectively, our data provide a comprehensive overview of the complex network of shapes, charges and interactions that enable LH3 glycosyltransferase activities, expanding the molecular framework for the manipulation of glycosyltransferase functions in biomedical and biotechnological applications.

biochemistry↗

bioRxiv2019

Polymorphism analyses and protein modelling inform on functional specialization of Piwi clade genes in the arboviral vector Aedes albopictus

Current knowledge of the piRNA pathway is based mainly on studies on the model organism Drosophila melanogaster, where three proteins of the Piwi subclade of the Argonaute family interact with PIWI-interacting RNAs to silence transposable elements in gonadal tissues. In mosquito species that transmit epidemic arboviruses such as the Dengue and Chikungunya viruses, Piwi clade genes underwent expansion, are also expressed in the soma, and code for proteins that may elicit antiviral functions and crosstalk with other proteins of recognized antiviral mechanisms. These observations underline the importance of expanding our knowledge of the piRNA pathway beyond D. melanogaster.\n\nHere we focus on the emerging arboviral vector Aedes albopictus and we couple traditional approaches of expression and adaptive evolution analyses with most current computational predictions of protein structure to study evolutionary divergence among Piwi clade proteins. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with several sites under positive selection. Expression profiles of Piwi transcripts during mosquito development and after infection with the Dengue 1 virus showed a concerted elicitation of all Piwi transcripts during viral dissemination, while the maintenance of infection primarily relied on the expression of Piwi5. In contrast, establishment of persistent infection by the Chikungunya virus is accompanied by an increased expression of all Piwi genes, particularly Piwi4 and, again, Piwi5. Overall these results are consistent with functional specialization and a general antiviral role for Piwi5. Experimental evidences of sites under positive selection in Piwi1/3, Piwi4 and Piwi6, further provide useful knowledge to design tailored functional experiments.\n\nAuthor summaryArgonautes are ancient proteins involved in many cellular processes, including innate immunity. Early in eukaryote evolution, Argonautes separated into Ago and Piwi clades, which maintain a dynamic evolutionary history with frequent duplications and losses. The use of Drosophila melanogaster as a model organism proved fundamental to understand the function of Argonautes. However, recent studies showed that the patterns and observations made in D. melanogaster, including the number of Argonautes, their expression profile and their function, are a rarity among Dipterans.\n\nIn vectors of epidemic arboviruses such as Dengue and Chikungunya viruses, Piwi genes underwent expansion, are expressed in the soma, and some of them appear to have antiviral functions. Besides being an important basic question, the identification of which (and how) Piwi genes have antiviral functions may be used for the development of novel genetic-based strategies of vector control. Here we coupled population genetics models with computational predictions of protein structure and expression analyses to investigate the evolution and function of Piwi genes of the emerging vector Aedes albopictus. Our data support a general antiviral role for Piwi5. Instead, the detection of complex expression profiles with the presence of sites under positive selection in Piwi1/3, Piwi4 and Piwi6 requires tailored functional experiments to clarify their antiviral role.

evolutionary biology↗