Biology subjects

Ricagno, S.

Publications and source records attributed to Ricagno, S..

16 recordsLinked to original sources

bioRxiv2025

Exploring light chain cardiotoxicity in AL amyloidosis: Impact on hiPSC-derived Cardiomyocyte Activity

AimsImmunoglobulin light chain (AL) amyloidosis is a protein misfolding disease characterized by the systemic deposition of amyloid fibrils derived from monoclonal light chains (LCs). Cardiac involvement is the major determinant of prognosis and mortality, and beyond fibril accumulation, soluble cardiotoxic LCs play a critical role in disease progression. While current in vivo models like C. elegans and murine systems have demonstrated LC toxicity, they lack human relevance or fail to capture soluble LC-induced cardiotoxicity. This study aimed to characterize the electrophysiological effects of cardiotoxic LCs on a human-relevant model using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and ResultsTwo amyloidogenic cardiotoxic LCs (H3 and H6) from AL patients and one non-cardiotoxic LC (M10) from a multiple myeloma patient were biophysically characterized and tested in hiPSC-CMs at clinically relevant concentrations. Electrophysiological recordings revealed that H3 and H6 significantly reduced spontaneous action potential (AP) firing frequency and maximal upstroke velocity (dV/dt) in hiPSC-CMs, indicating impaired excitability. H6 also shortened AP duration. H3 exposure led to a [~]40% reduction in peak sodium current density and altered inactivation kinetics of the L-type calcium current, without affecting major pacemaker or repolarizing potassium (IKr or IKs) currents. In contrast, M10 had no effect on any measured parameter, validating the models ability to discriminate toxic from non-toxic LCs. ConclusionThis study demonstrates that hiPSC-CMs provide a clinically relevant human model to investigate LC-induced cardiotoxicity. Cardiotoxic LCs exert distinct but converging electrophysiological impairments, including disruption of sodium and L-type calcium currents, contributing to reduced excitability and altered AP morphology. These findings provide mechanistic insights into AL amyloidosis-related cardiac dysfunction and establish a foundation for future therapeutic screening targeting soluble LC toxicity in a human context.

pathology↗

bioRxiv2025

Tau catalyzes amyloid-β aggregation in a fold-dependent manner

Interactions between amyloidogenic proteins are emerging as critical drivers of neurodegenerative diseases, yet the molecular mechanisms remain poorly understood. In Alzheimers disease (AD) and chronic traumatic encephalopathy (CTE), co-deposition of tau and amyloid-{beta} (A{beta}) leads to accelerated disease progression. Here, we investigated the direct interaction between A{beta} and tau, combining in vitro reconstruction, computational modeling, and in vivo models. We show that tau aggregates with AD paired helical filament (PHF) and CTE folds catalyze the primary nucleation of A{beta}42 in a fold-specific manner, through an enzyme-like kinetic with recognition mechanisms. CTE fibrils exhibit the highest catalytic activity, also constraining A{beta}42 polymorphism. PHF and CTE tau fibrils increase A{beta}42 toxicity in SH-SY5Y neuroblastoma cells and transgenic Caenorhabditis elegans. Catalytic heterotypic interactions between amyloidogenic proteins offer new insights into the pathological mechanisms of multiple proteinopathies. The mechanisms described here may guide the structure-based design of new therapeutic agents targeting specific amyloidogenic interactions.

biochemistry↗

bioRxiv2025

The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples

Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (KD), association rate (kon), and dissociation rate (ko,), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression mixtures. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the methods broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.

biophysics↗

bioRxiv2024

A conformational fingerprint for amyloidogenic light chains.

Immunoglobulin light chain amyloidosis (AL) and multiple myeloma (MM) both share the overproduction of a clonal light chain (LC). However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics (MD) simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

biophysics↗

bioRxiv2023

Endolysin B: A new archetype in M. tuberculosis treatment

So far it is thanks to antibiotics that illnesses, such as Tuberculosis (TB), are treatable. However, antimicrobial misuse combined with Mycobacterium tuberculosis phenotypic plasticity are nullifying the effects of the existing therapies. As a result, increasingly people are dying (one person dies of TB every 20 seconds), especially due to the rise of multi-and extensively drug-resistant strains. There is indeed a urgent need for new and more effective therapies, which should match the requirement of avoiding the rise in drug resistance. Among them, the use of bacteriophage - bacteria-restricted viruses - or simply phage lytic enzyme (i.e., endolysin) represents one of the most promising alternatives. All phages encode for the endolysin A (LysA), which degrades the bacteria cell wall, finally leading to the release of the newly synthesized virions. Nevertheless, mycobacteriophages (bacterial viruses selectively infecting mycobacteria), evolved the additional endolysin B (LysB) to selectively damage the complex mycobacteria cell wall, and to evade from their host. LysB, owing a lipolytic enzyme, can degrade the thick mycolic acid layer, and hence disrupt the integrity of the mycobacterial membrane. Despite its key role in mediating mycobacteria lysis, the molecular mechanism regulating LysB binding to its target remains poorly characterized. Herein, we selected Ms6LysB and created a fluorescent engineered version as a proxy to analyze LysB binding qualitatively and quantitatively to both the fast-growing non-pathogenic Mycobacterium smegmatis and the slow-growing pathogenic M. tuberculosis. Additionally, we shed light on LysB antimicrobial activity upon M. tuberculosis infection, by using alveolar-like mouse macrophages (mAMs) as a cellular model that closely recapitulates the natural niche of M. tuberculosis infection. Our study provides the proof-of-principle that Ms6 LysB binding to the outer mycobacterial membrane can impair M. tuberculosis growth homeostasis and that LysB retain its lytic properties even when internalized by mAMs. This lays the groundwork for the use of LysB as a new therapeutic strategy to undermine M. tuberculosis infection.

microbiology↗

bioRxiv2023

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

biophysics↗

bioRxiv2023

The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with chemokine CXCL12

Chemokines engage in heterodimeric interactions to activate or dampen their cognate receptors in inflammatory conditions. The chemokine CXCL12 forms with the alarmin HMGB1 a patho-physiologically relevant heterocomplex (HMGB1[bullet]CXCL12), whose formation synergically promotes the inflammatory response elicited by the G-protein coupled receptor CXCR4. However, the molecular details of complex formation were still elusive. Through an integrative structural approach (NMR, AUC, ITC, MST, SAXS) we show that HMGB1[bullet]CXCL12 represents the first fuzzy chemokines heterocomplex reported so far. HMGB1 and CXCL12 form a dynamic equimolar assembly, rather than involving one HMGB1 and two CXCL12 molecules as previously assumed, with structured and unstructured HMGB1 regions recognizing the dimerization surface of CXCL12. We uncover an unexpected role of the acidic intrinsically disordered region (IDR) in heterocomplex formation and provide the first evidence that the acidic IDR facilitates the ternary HMGB1*CXCL12*CXCR4 interaction on the cell surface. Thus, the interaction of HMGB1 with CXCL12 diverges radically from the classical rigid heterophilic chemokine-chemokine dimerization. Simultaneous interference with the multiple interactions within HMGB1[bullet]CXCL12 complex formation might offer novel pharmacological strategies to inhibit its detrimental activity in inflammatory conditions.

biochemistry↗

bioRxiv2022

Cryo-EM Structure of a Mammalian-specific Alternative Amyloid Exon

hnRNPDL is a ribonucleoprotein (RNP) involved in transcription and RNA-processing, with missense mutations causing limb-girdle muscular dystrophy-3 (LGMDD3). Mammalian-specific alternative splicing (AS) renders three natural isoforms, hnRNPDL-2 being predominant in humans. We present the cryo-electron microscopy structure of full-length hnRNPDL-2 amyloid fibrils, which are stable, non-toxic, and bind nucleic acids, with the RNA binding domains building a solenoidal coat around them. The amyloid core consists of a single Gly/Tyr-rich and highly hydrophilic filament containing internal water channels. The architecture and activity of hnRNPDL-2 fibrils are reminiscent of functional amyloids, our results suggesting that LGMDD3 might be a loss-of-function disease associated with impaired fibrillation. Strikingly, the fibril core matches exon 6, absent in the soluble hnRNPDL-3 isoform. This provides structural evidence for AS controlling hnRNPDL assembly by precisely including/skipping an amyloid exon, a mechanism that holds the potential to generate functional diversity in RNPs.

biochemistry↗

bioRxiv2022

Goat PRP14 (gPRP14) has xeno-antigenic properties and works as a vaccine in preclinical models of cancer.

We studied the activity of recombinant goat PRP14 (gPRP14), a member of the RID protein family, as a xeno-antigen in preclinical models of cancer. Antisera from rabbits and mice immunized with gPRP14 showed strong reactivity against several tumor cell types, which was absent towards normal cells: the tumor selectivity was related to surface and intra-cellular expression in tumor cells, and to an exclusively intra-cellular localization in normal cells. In vitro, binding to tumor cells was followed by cytotoxicity which could be rescued by the addition of excess soluble antigen. In vivo, an anti-tumor activity of immunization with gPRP14 was observed in murine syngeneic models of breast cancer and melanoma: the anti-tumor response was present when gPRP14 was administered in a preventive setting, and persisted upon repeated challenges with tumor cells in long-term survivor mice. Finally, we showed that both the humoral and T-cell mediated responses are needed for the optimal anti-tumor effect in the murine melanoma model. Thus, we have performed an initial characterization of gPRP14 as a cancer vaccine, which -given the potential wide range of tumor cells positive for the antigen-appears as a promising, novel immunotherapy.

cancer biology↗

bioRxiv2022

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-{beta} amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. The disease can be transmitted horizontally, likely facilitated by prion-like mechanism, in captive animals leading to extreme disease prevalence, e.g. 70% in captive cheetah and 57-73% in domestic short hair (DSH) cats kept in shelters. Herein, we present the 3.3 [A] cryo-EM structure of an AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure. The structure reveals a cross-{beta} architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Based on shared disease profiles and almost identical protein sequences, we propose a similar amyloid fold of deposits identified previously in captive cheetah.

biophysics↗

bioRxiv2022

AA-amyloidosis in cats (Felis catus) housed in shelters

Systemic AA-amyloidosis is a protein-misfolding disease that is characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. In domestic short-hair cats, AA-amyloidosis has only been anecdotally reported and is considered a rare disease. Here we report that an astonishing 57-73% of early deceased short-hair cats kept in three independent shelters suffer from amyloid deposition in the liver, spleen, or kidney. Histopathology and mass spectrometry of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B=0.026, CI95%=0.007-0.046; p=0.010). Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease.

pathology↗

bioRxiv2022

Multi-eGO: an in-silico lens to look into protein aggregation kinetics at atomic resolution

Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 diseases associated that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the characterization of the structures of the transient oligomers formed in the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long-time scales typical of aggregation processes have limited, so far, the use of computational methods. To surmount these limitations, we introduce here multi-eGO, an atomistic, hybrid structure-based model, which leveraging on the knowledge of monomers conformational dynamics and of fibril structures, can efficiently capture the essential structural and kinetics aspects of protein aggregation. Multi-eGO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments on an amyloidogenic peptide of Transthyretin, a protein responsible for one of the most common cardiac amyloidosis. Multi-eGO simulations allow to observe in time and at atomic resolution the formation of primary nuclei in a sea of transient lower order oligomers, to follow their growth and the subsequent secondary nucleation events, till the maturation of multiple fibrils. Multi-eGO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases. Significance StatementAlzheimers and Parkinsons diseases are uncurable pathologies associated to the aberrant aggregation of specific proteins into amyloid fibrils. Understanding the mechanism leading to protein aggregation, by characterizing the structures of the oligomeric species populated in the process, would have a tremendous impact on the design of therapeutic molecules. We propose that a structure-based approach to molecular dynamics simulations can allow following at high resolution the aggregation kinetics of thousands of monomers. Having shown that simulations can describe the aggregation of a Transthyretin amyloidogenic peptide, we demonstrate how their efficiency allows acquiring a wealth of structural information. We foresee that integrating the latter with the many techniques developed to study protein aggregation will support the design of molecules to modulate amyloidogenesis.

biochemistry↗

bioRxiv2021

Fragment-based computational design of antibodies targeting structured epitopes

De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of predetermined epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to pre-selected epitopes. summaryA combinatorial method can rapidly design nanobodies for predetermined epitopes, which bind with KDs in the nanomolar range.

bioinformatics↗

bioRxiv2020

Identification of variable sites in Sars-CoV-2 and their abundance profiles in time

The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.

bioinformatics↗

bioRxiv2020

Conformational stability and dynamics in crystals recapitulate protein behaviour in solution

A growing body of evidences has established that in many cases proteins may preserve most of their function and flexibility in a crystalline environment, and several techniques are today capable to detect transiently-populated states of macromolecules in tightly packed lattices. Intriguingly, in the case of amyloidogenic precursors, the presence of these conformations (hidden to conventional crystallographic studies) can be correlated to the pathological fate of the native fold. It remains unclear, however, to which extent these minor conformations reflect the protein behaviour that is more commonly studied in solution. Here, we address this question by investigating some biophysical properties of a prototypical amyloidogenic system, {beta}2-microglobulin ({beta}2m) in solution and in microcrystalline state. By combining NMR chemical shifts with Molecular Dynamics (MD) simulations, we confirmed that conformational dynamics of {beta}2m native state in the crystal lattice is in keeping with what observed in solution. A comparative study of protein stability in solution and in crystallo is then carried out, monitoring the change in protein secondary structure at increasing temperature by Fourier transform infrared (FTIR) spectroscopy. The increased structural order of the crystalline state contributes to provide better resolved spectral components compared to those collected in solution and crucially, the crystalline samples display thermal stabilities in good agreement with the trend observed in solution. Overall, this work shows that protein stability and occurrence of pathological hidden states in crystals parallel their solution counterpart, confirming the interest of crystals as a platform for the biophysical characterisation of processes such as unfolding and aggregation.

biochemistry↗

bioRxiv2018

Cryo-EM structure of cardiac amyloid fibrils from an immunoglobulin light chain (AL) amyloidosis patient

Systemic light chain (AL) amyloidosis is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 [A] resolution cryo-electron microscopy (cryo-EM) map and structural model of amyloid fibrils extracted from the heart of an AL patient affected by severe amyloid cardiomyopathy. The fibrils are composed of one asymmetric protofilament, showing typical 4.9 [A] stacking and parallel cross-{beta} architecture. Two distinct polypeptide stretches belonging to the LC variable domain (Vl) could be modelled in the density (total of 77 residues), stressing the role of the Vl domain in fibril assembly and LC aggregation. Despite high levels of Vl sequence variability, residues stabilising the observed fibril core are conserved through several Vl domains, highlighting structural motifs that may be common to misfolded LCs. Our data shed first light on the architecture of life-threatening LC amyloid deposits, and provide a rationale for correlating LC amino acid sequences and fibril structures.

biochemistry↗