Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.     

Prediction of Quality-control Degradation Signals in Yeast Proteins

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin–proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.     

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of ⁵²PPQ⁵⁴ residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the ⁵²PPQ⁵⁴ region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.     

Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein using Hydrogen Exchange-Mass Spectrometry

Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N ↔ I₁ ↔ I₂ ↔ I₃ ↔ U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins.     

Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.     

TAPASS: Tool for annotation of protein amyloidogenicity in the context of other structural states

Numerous studies have demonstrated that the propensity of a protein to form amyloids or amorphous aggregates is encoded by its amino acid sequence. This led to the emergence of several computational programs to predict amyloidogenicity from amino acid sequences. However, a growing number of studies indicate that an accurate prediction of the protein aggregation can only be achieved when also accounting for the overall structural context of the protein, and the likelihood of transition between the initial state and the aggregate. Here, we describe a computational pipeline called TAPASS, which was designed to do just that. The pipeline assigns each residue of a protein as belonging to a structured region or an intrinsically disordered region (IDR). For this purpose, TAPASS uses either several state-of-the-art programs for prediction of IDRs, of transmembrane regions and of structured domains or the artificial intelligence program AlphaFold. In the next step, this assignment is crossed with amyloidogenicity prediction. As a result, TAPASS allows the detection of Exposed Amyloidogenic Regions (EARs) located within intrinsically disordered regions (IDRs) and carrying high amyloidogenic potential. TAPASS can substantially improve the prediction of amyloids and be used in proteome-wide analysis to discover new amyloid-forming proteins. Its results, combined with clinical data, can create individual risk profiles for different amyloidoses, opening up new opportunities for personalised medicine. The architecture of the pipeline is designed so that it makes it easy to add new individual predictors as they become available. TAPASS can be used through the web interface (https://bioinfo.crbm.cnrs.fr/index.php?route=tools&tool=32).     

Structural Studies Reveal Unique Non-canonical Regulators of G Protein Signaling Homology (RH) Domains in Sorting Nexins

As a subgroup of sorting nexins (SNXs) that contain regulator of G protein signaling homology (RH) domain, SNX-RH proteins, including SNX13, SNX14 and SNX25, were proposed to play bifunctional roles in protein sorting and GPCR signaling regulation. However, mechanistic details of SNX-RH proteins functioning via RH domain remain to be illustrated. Here, we delineate crystal structures of the RH domains of SNX13 and SNX25, revealing a homodimer of SNX13 RH domain mediated by unique extended α4 and α5 helices, and a thiol modulated homodimer of SNX25-RH triggered by a unique cysteine on α6 helix. Further studies showed that RH domains of SNX-RH do not possess binding capacity toward Gα subunits, owing to the lack of critical residues for interaction. Thus, this study identifies a group of novel non-canonical RH domains that can act as a dimerization module in sorting nexins, which provides structural basis for mechanism studies on SNX-RH protein functions.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator

We have previously shown that the CBb subunit of crotoxin, a β-neurotoxin with phospholipase A₂ (PLA₂) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA₂ acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell.Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A₂ on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein–protein interactions.     

Extending the Horizon of Homology Detection with Coevolution-based Structure Prediction

Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a “beads-on-a-string” model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Structural Modeling of the SARS-CoV-2 Spike/Human ACE2 Complex Interface can Identify High-Affinity Variants Associated with Increased Transmissibility

The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are structurally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S477N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S477N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.     

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.     

Limited Invasive Protocol: Optimizing Diagnostic Modalities in Corticotropin Mediated Cushing Syndrome

BackgroundTo limit the role of bilateral inferior petrosal sinus sampling (BIPSS) in distinguishing between Cushing disease (CD) and ectopic Cushing syndrome (ECS), recent reports have proposed a noninvasive approach based on a combination of biochemical testing and radiological imaging as an alternative to the conventional invasive strategy (CIS). However, this strategy requires further validation. The current study aimed to evaluate 2 limited invasive protocols (LIP-1 and LIP-2) in limiting the role of BIPSS while maintaining a diagnostic accuracy similar to that of CIS.MethodsThis was a single-center study conducted on individuals with corticotropin-dependent Cushing syndrome. The LIPs were based on performing high-dose dexamethasone suppression (>50% cut-off in first [LIP-1] and >80% in second [LIP-2]) and magnetic resonance imaging of the sella in all individuals and selective use of computed tomography of the chest and abdomen before BIPSS. These LIPs were evaluated for limiting the use of BIPSS, their accuracy, and cost in comparison to CIS.ResultsOf the 206 individuals, 114 (97 of CD and 21 of ECS) were eligible for the current study. Using LIP-1, LIP-2, and CIS, BIPSS could have been avoided in 62.3%, 35.9%, and 25.4% of individuals, respectively. The positive predictive value for CD using LIP-1 and LIP-2 was 98.9% and 100%, respectively. The cost per patient evaluated using LIP-1, LIP-2, and CIS was $602.21, $966.81, and $1107.78, respectively.ConclusionLIPs represent an equally accurate, less invasive, and more cost-effective alternative to the CIS for distinguishing between CD and ECS.     

Revisiting misfolding propensity of serum amyloid A1: Special focus on the signal peptide region

AA amyloidosis is the result of overproduction and aberrant processing of acute-phase serum amyloid A1 (SAA1) by hepatocytes. Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein undergoes a cleavage of 18 residues consisting of the signal peptide at the N-terminal region. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region. We first examined the signal peptide amino acid SAA derived from different animal species. A library of 16 peptides was designed to evaluate the propensity of aggregation. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using in silico Tango program, thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and seeding with misfolded human SAA1 signal peptide. After 7 days of incubation, most of the SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 100 μM in 50 mM Tris buffer at 37 °C by TEM. All peptides were able to generate fibrils at a higher concentration, i.e 500 μM in 25 mM Tris buffer with 50% HFIP, by ThT. All SAA1 signal synthetic peptides designed from the different animal species had the propensity to misfold and form fibrils, particularly in species with low occurrence of systemic amyloidosis. The human SAA1 signal peptide region was capable to seed the SAA1 1–25 and 32–47 peptide regions. Characterizing fibrillar conformations are relevant for seeding intact and/or fragmented SAA, which may contribute, to the mechanism of protein misfolding. This research signifies the importance of the signal peptide region and its possible contribution to the misfolding of aggregation-prone proteins.     

Missense Variants Reveal Functional Insights Into the Human ARID Family of Gene Regulators

Missense variants are alterations to protein coding sequences that result in amino acid substitutions. They can be deleterious if the amino acid is required for maintaining structure or/and function, but are likely to be tolerated at other sites. Consequently, missense variation within a healthy population can mirror the effects of negative selection on protein structure and function, such that functional sites on proteins are often depleted of missense variants. Advances in high-throughput sequencing have dramatically increased the sample size of available human variation data, allowing for population-wide analysis of selective pressures. In this study, we developed a convenient set of tools, called 1D-to-3D, for visualizing the positions of missense variants on protein sequences and structures. We used these tools to characterize human homologues of the ARID family of gene regulators. ARID family members are implicated in multiple cancer types, developmental disorders, and immunological diseases but current understanding of their mechanistic roles is incomplete. Combined with phylogenetic and structural analyses, our approach allowed us to characterise sites important for protein-protein interactions, histone modification recognition, and DNA binding by the ARID proteins. We find that comparing missense depletion patterns among paralogs can reveal sub-functionalization at the level of domains. We propose that visualizing missense variants and their depletion on structures can serve as a valuable tool for complementing evolutionary and experimental findings.