Biology subjects

Spagnolli, G.

Publications and source records attributed to Spagnolli, G..

10 recordsLinked to original sources

bioRxiv2026

Protein Folding Interference Enables Selective Cyclin D1 Degradation

Cyclin D1 is a central regulator of cell-cycle progression and a compelling therapeutic target in cancer, yet its non-enzymatic function, limited ligandability, and rapid turnover have hindered direct pharmacological intervention. Here we show that these limitations can be overcome by targeting a transient conformational state populated during protein folding. Computational reconstruction of the Cyclin D1 folding landscape enabled the identification of a ligandable folding intermediate that served as the basis for structure-guided discovery of small molecules inducing selective degradation of nascent Cyclin D1. Orthogonal computational, biophysical, and cellular studies support the proposed mechanism, linking engagement of the folding intermediate to selective degradation of newly synthesized Cyclin D1. Pharmacological folding interference suppressed Cyclin D1 signaling, inhibited tumor cell proliferation, produced antitumor activity in vivo, and retained efficacy in a model of acquired resistance to CDK4/6 inhibition. Together, our findings establish a rational strategy for directly targeting Cyclin D1. More broadly, they suggest that the conformational landscape explored during protein folding represents an untapped reservoir of therapeutic opportunities, expanding small-molecule discovery beyond the native structures that have traditionally defined the druggable proteome.

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

Long range allostery mediates the regulation of plasminogen activator inhibitor 1 by vitronectin

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable, active conformation, with a solvent accessible reactive center loop (RCL), to a stable, inactive or latent conformation in which the RCL has inserted into the central {beta} sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin which retards the rate of this latency transition approximately 2-fold. We investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central {beta} sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition, and this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 [A] away from the binding site thus blocking the transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that have previously been identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin bound state which corresponds to a cryptic drug binding site identified by crystallography. Overall, these results shed new light on regulation of the PAI-1 latency transition by vitronectin and illustrate the potential of path sampling simulations for understanding functional conformational changes in proteins and for facilitating drug discovery.

biophysics↗

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

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

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↗