Biology subjects

Biasini, E.

Publications and source records attributed to Biasini, E..

13 recordsLinked to original sources

bioRxiv2026

Loss of the Central Region Reshapes the Dynamic Landscape of the Cellular Prion Protein and Its Plasma Membrane Interaction

Prion diseases are fatal neurodegenerative disorders driven by the conversion of the cellular prion protein (PrP) into a misfolded, pathogenic conformer. Beyond serving as a substrate for prion propagation, PrP is also thought to mediate neurotoxic signaling. Within this framework, the central region of PrP has emerged as a critical regulatory element. Notably, deletion of residues 105-125 ({Delta}CR) leads to spontaneous neurodegeneration in vivo and induces abnormal ionic currents in cultured cells and primary neurons, indicating that this region is essential for controlling the toxicity of the N-terminal domain. Current models propose that the N-terminus functions as a toxic effector whose activity is modulated by the C-terminal domain. This intramolecular interplay is likely central to the physiological role of PrP, and its disruption may contribute to neurodegeneration. Here, we investigated how deletion of the central region affects the structure and dynamics of full-length PrP. We generated membrane-bound models of full-length, diglycosylated wild-type (WT) PrP and the neurotoxic {Delta}CR mutant, and compared their conformational dynamics using molecular dynamics simulations. The two proteins exhibited markedly distinct behaviours. WT PrP adopted a more compact conformational ensemble of the N-terminal domain, consistent with stabilizing interactions between the flexible N-terminus and the globular C-terminal domain. In contrast, the {Delta}CR variant displayed more extended conformations and a substantial redistribution of intramolecular contacts, including the loss of specific interactions between the disordered N-terminal tail and the globular domain. This altered structural organization was accompanied by an increased propensity of the N-terminal domain to approach the membrane surface in the mutant. Our results provide a molecular model in which the central region engages intramolecular interaction networks that ultimately help regulate N-terminal residence at the plasma membrane, offering mechanistic insight into how CR deletion shifts the conformational ensemble toward membrane-associated states that may be associated with neurotoxic activity.

biochemistry↗

bioRxiv2025

Unfold to refold: Tracking the initial steps of PrPC unfolding in the context of PrPSc propagation

It might have been believed that elucidation of the atomistic structure of PrPSc would lead to an immediate understanding of the mechanism of prion propagation. However, PrPSc, now known to be a "simple" amyloid, can just template a previously unfolded polypeptide chain. Therefore, PrPSc can easily template the disordered [~]90-120 domain of an incoming PrPC molecule, but not its [~]121-231 folded domain (FD). The FD needs to accommodate into the [~]121-230 PrPSc surface, an inert "procrustean bed". Thus, a mechanism for concerted unfolding/refolding of the FD must exist, with FD unfolding as a key element. To explore how this might happen, we performed thermal unfolding of recombinant bank vole PrPC(90-231), that is a universal PrPSc propagator PrPSc, tracking changes at the residue level with solution NMR to pinpoint early unfolding propensity. Our data suggest that a key early event is the destabilization of the short {beta}1-{beta}2 assembly, and that the segment contiguous to the disordered tail, [~]121-140, encompassing {beta}1 and its adjacent coils, is the most likely region to unfold first. Spectroscopic data obtained at higher temperatures suggest that portions of alpha helix 2 are likely the last elements of the FD to unfold and refold into the PrPSc conformation. Molecular Dynamics simulations assisted the interpretation of these changes and suggest separation of 1 from the rest of the FD ensemble. Our data provide a conceivable timeline of the early events in PrPSc-assisted conversion of PrPC and should serve as a starting framework to develop a future atomistic model of PrPSc propagation.

biochemistry↗

bioRxiv2024

Mapping Cryptic Phosphorylation Sites in the Human Proteome

Advances in computational and experimental methods have revealed the existence of transient, non-native protein folding intermediates that could play roles in disparate biological processes, from regulation of protein expression to disease-relevant misfolding mechanisms. Here, we tested the possibility that specific post-translational modifications may involve residues exposed during the folding process by assessing the solvent accessibility of 87,138 post-translationally modified amino acids in the human proteome. Unexpectedly, we found that one-third of phosphorylated proteins present at least one phosphosite completely buried within the proteins inner core. Computational and experimental analyses suggest that these cryptic phosphosites may become exposed during the folding process, where their modification could destabilize native structures and trigger protein degradation. Phylogenetic investigation also reveals that cryptic phosphosites are more conserved than surface-exposed phosphorylated residues. Finally, cross-referencing with cancer mutation databases suggests that phosphomimetic mutations in cryptic phosphosites can increase tumor fitness by inactivating specific onco-suppressors. These findings define a novel role for co-translational phosphorylation in shaping protein folding and expression, laying the groundwork for exploring the implications of cryptic phosphosites in health and disease.

biochemistry↗

bioRxiv2022

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 WY conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 {micro}M binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 {micro}M in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan. Significance StatementWhen a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell - availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.

biochemistry↗

bioRxiv2022

Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency

The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation occurring in the population of south Brazil. In this work, we have combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. Besides the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt bridge interaction that stabilizes the p53 tetramerization domain were studied (R337D, D352R, and the double mutation R337D plus D352R). Simulations revealed a destabilizing effect of pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay performed in yeast by tuning the expression of wild-type and mutant p53 proteins revealed that p53 tetramerization mutations could decrease transactivation potential and alter transactivation specificity, in particular, by better tolerating the negative features in weak DNA binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53 conformational stability and function.

cancer biology↗

bioRxiv2021

Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials. Author SummaryThe pandemic caused by the SARS-CoV-2 virus has created a completely unusual situation in rapidly searching for compounds able to interfere with the biological processes exploited by the virus. This new scenario has substantially changed the timing of drug development which has also resulted in the generation of controversial results, proving that the transition from computational screening to the clinical application requires great caution and careful studies. It is therefore necessary to establish new paradigms for evaluating the efficacy of a potential active molecule. We set up a preclinical platform aimed at identifying molecules active against SARS-CoV-2 infection developing a multidisciplinary approach based on very stringent experimental criteria, comprising in-silico studies, in vitro binding tests and infection studies with pseudovirus expressing the spike protein as well as clinically isolated SARS-CoV-2 strains. We focused our attention on doxycycline which has been suggested as potential therapeutic candidate for treating COVID-19 and is currently employed in about twenty clinical trials. Doxycycline resulted effective in inhibiting the transduction of pseudovirus but it did not affect the entry and replication of SARS-CoV-2. The results obtained underline the need to define more stringent and controlled pharmacological approaches before wasting precious time and resources with clinical trials.

pharmacology and toxicology↗

bioRxiv2021

Solid state NMR reveals a parallel in register architecture for an infectious recombinant prion

Two alternative architectures have been proposed for PrPSc, the most notorious prion: a parallel in register {beta} stack (PIRIBS) and a 4-rung {beta}-solenoid (4R{beta}S). We challenged these two models by measuring intermolecular 13C-13C dipole-dipole couplings of 13CO-labelled Phe residues in a fully infectious sample of recombinant bank vole PrPSc (recBVPrPSc) using a PITHIRDS-CT solid state NMR (ssNMR) experiment. To our surprise, data strongly support a PIRIBS architecture. However, the mean distance measured ([~]6.5 [A]) suggests that a minimum of two of the three Phe residues are not perfectly stacked at the canonical [~]5 [A] cross-{beta} distance. Additional ssNMR experiments show some local conformational variability of the Phe residues within limits of a relatively high rigidity. The most parsimonious interpretation of our data is that recBVPrPSc is arranged as a PIRIBS, although additional conformers with alternative architectures cannot be excluded, including a mixture of PIRIBS and 4R{beta}S. Author summaryPrPSc is the most notorious prion. It is an infectious protein that cuases fatal neurodegenerative diseases in humans and animals. PrPSc is the aberrant version of a brain protein, PrPC. PrPSc and PrPC have the same prinary structure, but different secondary, tertiaty and quaternary structures. PrPSc is capable of templating PrPC to convert to the PrPSc conformation, which is the basis of its capacity to propagate. Two plausible structural models of PrPSc have been proposed, the four-rung {beta}-solenoid (4R{beta}S) and the parallel in-register {beta} stack (PIRIBS) model. In both cases the driving force of the templating mechanism consists of "sticky" surface {beta}-strands; however, in the PIRIBS model all the {beta}-strands that conform a PrPSc monomer lie flat on a surface whereas in the 4R{beta}S model they wind in a corkscrew fashion. Here, we analyzed fully infectious recombinant PrPSc using a solid state NMR technique, PITHIRDS, that allows probing distances between specific labelled amino acid residues. To our surprise (as we have defended the 4R{beta}S model in the past), results clearly show the presence of a PIRIBS structure in our sample.

biochemistry↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Expression and protein sequence analyses of zebrafish impg2a and impg2b, two proteoglycans of the interphotoreceptor matrix

Photoreceptor outer segments projecting from the surface of the neural retina toward the retinal pigment epithelium (RPE) are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix (IPM) [1,2]. This extracellular compartment is necessary for physiological retinal function. However, specific roles for molecules characterizing the IPM have not been clearly defined [3]. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene in patients affected by autosomal recessive Retinitis Pigmentosa (arRP) [4,5] and autosomal dominant and recessive vitelliform macular dystrophy (VMD) [6,7]. The gene encodes for a proteoglycan synthesized by photoreceptors and secreted in the IPM. However, little is known about the function and structure of this protein. We used the teleost zebrafish (D.rerio) as a model to study IMPG2 expression both during development and in adulthood, as its retina is very similar in humans [8]. In zebrafish, there are two IMPG2 proteins, IMPG2a and IMPG2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among different vertebrate species, showing a significant similarity despite the evolutionary distance between humans and teleosts. In fact, human IMPG2 and D.rerio IMPG2a and IMPG2b share conserved SEA and EGF-like domains. Homology models of these domains were obtained by using the iTasser server. Finally, expression analyses of impg2a and impg2b during development and in the adult fish showed expression of both mRNAs starting from 3 days post fertilization (dpf) in the outer nuclear layer of zebrafish retina that continues throughout adulthood. This data lays the groundwork for the generation of novel and most needed animal models for the study of IMPG2-related inherited retinal dystrophies.

developmental biology↗

bioRxiv2020

All-Atom Simulation of the HET-s Prion Replication

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. In this work, we focus on the propagation of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Since time-resolved biophysical experiments cannot yield a full reconstruction of prion replication, we resort to computational methods. To overcome the computational limitations of plain Molecular Dynamics (MD) simulations, we adopt a special type of biased dynamics called ratchet-and-pawl MD (rMD). The accuracy of this enhanced path sampling protocol strongly depends on the choice of the collective variable (CV) used to define the biasing force. Since for prion propagation a reliable reaction coordinate (RC) is not yet available, we resort to the recently developed Self-Consistent Path Sampling (SCPS). Indeed, in such an approach the CV where the biasing force is applied is not heuristically postulated but is calculated through an iterative refinement procedure. Our atomistic reconstruction of the HET-s replication shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained with a different computational protocol. Together, these results indicate that the propagation of prions generated by evolutionary distant proteins shares common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.

biophysics↗

bioRxiv2020

Structural features of an infectious recombinant PrPSc prion using solid state NMR

PrPSc, the first described and most notorious prion, is the only protein known to cause epidemics of deadly disease. Its properties are encoded in its unique structure. Here we report a first solid state NMR study of a uniformly labelled (U-13C,15N)-Bank vole (BV) infectious recombinant PrPSc prion. C-C, C-H and N-H spectra were obtained with MAS rotation of the sample at up to 60 kHz. We obtained amino acid-type secondary structure information and used it to challenge a physically plausible atomistic model of PrPSc consisting of a 4-rung {beta} solenoid recently proposed by us. In all cases, our model was compatible with the data. This study shows that elucidation of the structure of PrPSc is within reach using recombinant PrPSc, NMR, and our model as a guiding tool.

biochemistry↗

bioRxiv2020

PHARMACOLOGICAL PROTEIN INACTIVATION BY TARGETING FOLDING INTERMEDIATES

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

biochemistry↗

bioRxiv2018

Full Atomistic Model Of Prion Structure And Conversion

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), is the only one known to cause epidemic and epizootic episodes(1). Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-{beta}-Sheet (PIRIBS)(2) or {beta}-solenoid conformations(3). Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung {beta}-solenoid (4R{beta}S)(4, 5). Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4R{beta}S architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring {beta}-solenoid. Importantly, the 4R{beta}S arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. Our results provide the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.\n\nSignificanceSince the original hypothesis by Stanley Prusiner, prions have represented enigmatic agents diverging from the classical concept of genetic inheritance. However, the structure of PrPSc, the infectious isoform of the cellular prion protein (PrPC), has so far remained elusive, mostly due to technical challenges posed by its aggregation propensity. Here, we present a new high resolution model of PrPSc derived from the integration of a wide array of recent experimental constraints. By coupling the information of such model with a newly developed computational method, we reconstructed for the first time the conformational transition of PrPC to PrPSc. This study offers a unique workbench for designing therapeutics against prion diseases, and a physically-plausible mechanism explaining how protein conformation could self-propagate.

biochemistry↗