Pliability in the m6A-Binding Region Extends Druggability of YTH Domains.

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

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

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, 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. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 "WY" conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 µM binding affinity for CtUGGTGT24in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 µM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors.

Gene Expression Profiling in Trigeminal Ganglia from Cntnap2-/- and Shank3b-/- Mouse Models of Autism Spectrum Disorder.

Sensory difficulties represent a crucial issue in the life of autistic individuals. The diagnostic and statistical manual of mental disorders describes both hyper- and hypo-responsiveness to sensory stimulation as a criterion for the diagnosis autism spectrum disorders (ASD). Among the sensory domain affected in ASD, altered responses to tactile stimulation represent the most commonly reported sensory deficits. Although tactile abnormalities have been reported in monogenic cohorts of patients and genetic mouse models of ASD, the underlying mechanisms are still unknown. Traditionally, autism research has focused on the central nervous system as the target to infer the neurobiological bases of such tactile abnormalities. Nonetheless, the peripheral nervous system represents the initial site of processing of sensory information and a potential site of dysfunction in the sensory cascade. Here we investigated the gene expression deregulation in the trigeminal ganglion (which directly receives tactile information from whiskers) in two genetic models of syndromic autism (Shank3b and Cntnap2 mutant mice) at both adult and juvenile ages. We found several neuronal and non-neuronal markers involved in inhibitory, excitatory, neuroinflammatory and sensory neurotransmission to be differentially regulated within the trigeminal ganglia of both adult and juvenile Shank3b and Cntnap2 mutant mice. These results may help in disentangling the multifaced complexity of sensory abnormalities in autism and open avenues for the development of peripherally targeted treatments for tactile sensory deficits exhibited in ASD.

Functional, pathogenic, and pharmacological roles of protein folding intermediates.

Protein expression and function in eukaryotic cells are tightly harmonized processes modulated by the combination of different layers of regulation, including transcription, processing, stability, and translation of messenger RNA, as well as assembly, maturation, sorting, recycling, and degradation of polypeptides. Integrating all these pathways and the protein quality control machinery, deputed to avoid the production and accumulation of aberrantly folded proteins, determines protein homeostasis. Over the last decade, the combined development of accurate time-resolved experimental techniques and efficient computer simulations has opened the possibility of investigating biological mechanisms at atomic resolution with physics-based models. A meaningful example is the reconstruction of protein folding pathways at atomic resolution, which has enabled the characterization of the folding kinetics of biologically relevant globular proteins consisting of a few hundred amino acids. Combining these innovative computational technologies with rigorous experimental approaches reveals the existence of non-native metastable states transiently appearing along the folding process of such proteins. Here, we review the primary evidence indicating that these protein folding intermediates could play roles in disparate biological processes, from the posttranslational regulation of protein expression to disease-relevant protein misfolding mechanisms. Finally, we discuss how the information encoded into protein folding pathways could be exploited to design an entirely new generation of pharmacological agents capable of promoting the selective degradation of protein targets.

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 present in the population in south Brazil. In this work, we 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. In addition to 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 (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the 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 the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53's conformational stability and function.

Defective cyclophilin A induces TDP-43 proteinopathy: implications for amyotrophic lateral sclerosis and frontotemporal dementia.

Aggregation and cytoplasmic mislocalization of TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis and frontotemporal dementia spectrum. However, the molecular mechanism by which TDP-43 aggregates form and cause neurodegeneration remains poorly understood. Cyclophilin A, also known as peptidyl-prolyl cis-trans isomerase A (PPIA), is a foldase and molecular chaperone. We previously found that PPIA interacts with TDP-43 and governs some of its functions, and its deficiency accelerates disease in a mouse model of amyotrophic lateral sclerosis. Here we characterized PPIA knock-out mice throughout their lifespan and found that they develop a neurodegenerative disease with key behavioural features of frontotemporal dementia, marked TDP-43 pathology and late-onset motor dysfunction. In the mouse brain, deficient PPIA induces mislocalization and aggregation of the GTP-binding nuclear protein Ran, a PPIA interactor and a master regulator of nucleocytoplasmic transport, also for TDP-43. Moreover, in absence of PPIA, TDP-43 autoregulation is perturbed and TDP-43 and proteins involved in synaptic function are downregulated, leading to impairment of synaptic plasticity. Finally, we found that PPIA was downregulated in several patients with amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia, and identified a PPIA loss-of-function mutation in a patient with sporadic amyotrophic lateral sclerosis . The mutant PPIA has low stability, altered structure and impaired interaction with TDP-43. These findings strongly implicate that defective PPIA function causes TDP-43 mislocalization and dysfunction and should be considered in future therapeutic approaches.

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

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.

Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention.

Memory consolidation requires astrocytic microdomains for protein recycling; but whether this lays a mechanistic foundation for long-term information storage remains enigmatic. Here we demonstrate that persistent synaptic strengthening invited astrocytic microdomains to convert initially internalized (pro)-brain-derived neurotrophic factor (proBDNF) into active prodomain (BDNFpro) and mature BDNF (mBDNF) for synaptic re-use. While mBDNF activates TrkB, we uncovered a previously unsuspected function for the cleaved BDNFpro, which increases TrkB/SorCS2 receptor complex at post-synaptic sites. Astrocytic BDNFpro release reinforced TrkB phosphorylation to sustain long-term synaptic potentiation and to retain memory in the novel object recognition behavioral test. Thus, the switch from one inactive state to a multi-functional one of the proBDNF provides post-synaptic changes that survive the initial activation. This molecular asset confines local information storage in astrocytic microdomains to selectively support memory circuits.

Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds 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 which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

Pharmacological inactivation of the prion protein by targeting a folding intermediate.

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. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels 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 load 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.

The Compelling Demand for an Effective PrPC-Directed Therapy against Prion Diseases.

Decades of research efforts have conclusively provided overwhelming evidence that the cellular prion protein (PrPC) plays a central role in prion diseases, a set of fatal and incurable neurodegenerative disorders for which no therapy is yet available. In this Viewpoint, we provide an overview of the drug discovery strategies in the field, highlighting the current therapeutic hypotheses targeting, whether directly or indirectly, PrPC as well as the antiprion agents closest to clinical application.

Generation, optimization and characterization of novel anti-prion compounds.

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.

Modeling PrPSc Generation Through Deformed Templating.

Deformed templating is the process by which self-replicating protein conformations with a given cross-ß folding pattern can seed formation of an alternative self-replicating state with different cross-ß folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a bona fide infectious conformer, PrPSc through deformed templating. The process can take many rounds of replication (if taking place in vitro) or even several passages of the evolving PrP conformers through successive brains if in vivo, through experimental transmission. In all cases, deformed templating involves a forced conversion in which there is a mismatch between the template and the substrate and/or the templating environment, typically a recombinant PrP amyloid, adept at converting recombinant PrP under denaturing conditions (e.g., presence of chaotropic agents), encountering a glycosylated, GPI-anchored PrPC substrate under physiological conversion conditions. Deformed templating is characterized by emergence of intermediate conformers that exhibit biochemical characteristics that are intermediate between those of the initial PrP amyloid and the final PrPSc conformers. Here, we took advantage of the recent elucidation of the structure of a PrP amyloid by cryo-EM and the availability of a physically plausible atomistic model of PrPSc that we have recently proposed. Using modeling and Molecular Dynamics (MD) approaches, we built a complete molecular modelization of deformed templating, including an atomistic model of a glycosylated intermediate conformer and a modified model of PrPSc. Among other unanticipated outcomes, our results show that fully glycosylated PrP can be stacked in-register, and how 4-rung ß-solenoid (4RßS) PrP architectures can share key structural motifs with parallel-in register intermolecular sheet (PIRIBS) PrP amyloids. Our results shed light on the mechanisms of prion replication.

Understanding prion structure and conversion.

Since their original identification, prions have represented enigmatic agents that defy the classical concept of genetic inheritance. For almost four decades, the high-resolution structure of PrPSc, the infectious and misfolded counterpart of the cellular prion protein (PrPC), has remained elusive, mostly due to technical challenges posed by its high insolubility and aggregation propensity. As a result, such a lack of information has critically hampered the search for an effective therapy against prion diseases. Nevertheless, multiple attempts to get insights into the structure of PrPSc have provided important experimental constraints that, despite being at limited resolution, are paving the way for the application of computer-aided technologies to model the three-dimensional architecture of prions and their templated replication mechanism. Here, we review the most relevant studies carried out so far to elucidate the conformation of infectious PrPSc and offer an overview of the most advanced molecular models to explain prion structure and conversion.

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. Since time-resolved biophysical experiments are currently unable to provide full reconstruction of the physico-chemical mechanisms responsible for prion replication, one must rely on computer simulations. In this work, we show that a recently developed algorithm called Self-Consistent Path Sampling (SCPS) overcomes the computational limitations of plain MD and provides a viable tool to investigate prion replication processes using state-of-the-art all-atom force fields in explicit solvent. First, we validate the reliability of SCPS simulations by characterizing the folding of a class of small proteins and comparing against the results of plain MD simulations. Next, we use SCPS to investigate the replication of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Our atomistic reconstruction shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained using a simpler and more approximate path sampling algorithm. Together, these results suggest that the propagation of prions generated by evolutionary distant proteins may share common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.

The cellular prion protein beyond prion diseases.

The cellular prion protein (PrPC), a cell surface glycoprotein originally identified for its central role in prion diseases (also called transmissible spongiform encephalopathies), has recently been implicated in the pathogenesis of other neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, by acting as a toxicity-transducing receptor for different misfolded protein isoforms, or in some case by exerting neuroprotective effects. Interestingly, PrPC has also been reported to play unexpected functions outside the nervous system, for example by contributing to myelin homeostasis, regulating specific processes of the immune system and participating in various aspects of cancer progression. Collectively, these observations point to a much broader role for PrPC in physiological and disease processes than originally assumed. In this manuscript, we provide an overview of what is known about the role of PrPC beyond prion disorders and discuss the potential implications of targeting this protein in different diseases.

Characterization of Physical, Mechanical, and Biological Properties of SilkBridge Nerve Conduit after Enzymatic Hydrolysis.

The in vitro degradation profile and the cytotoxicity of the degradation products of a silk fibroin (SF)-based nerve conduit (SilkBridge), with a complex three-layered wall architecture comprising both native and regenerated (electrospun) fibers, are reported. The bacterial protease type XIV from Streptomyces griseus was used as a hydrolytic agent at three different enzyme/substrate ratios (1:8, 1:80, and 1:800 w/w) to account for the different susceptibility to degradation of the native and regenerated components. The incubation time was extended up to 91 days. At fixed time points, the remaining device, the insoluble debris, and the incubation buffers containing soluble degradation products were separated and analyzed. The electrospun fibers forming the inner and outer layers of the conduit wall were almost completely degraded within 10 days of incubation at an enzyme/substrate ratio of 1:80 w/w. The progression of degradation was highlighted by the emergence of zones of erosion and discontinuity along the electrospun fibers, weakening of the electrospun layers, and decrease in resistance to compressive stress. Native SF microfibers forming the middle layer of the conduit wall displayed a higher resistance to enzymatic degradation. When incubated at an enzyme/substrate ratio of 1:8 w/w, the weight decreased gradually over the incubation time as a consequence of fiber erosion and fragmentation. Analogously, the tensile properties markedly decreased. Both spectroscopic and thermal analyses confirmed the gradual increase in the crystalline character of the fibers. The incubation buffers containing the soluble degradation products were subjected to cytotoxicity testing with human HEK293 cells and mouse neuroblastoma N2a cells. No detrimental effects on cell viability were observed, suggesting that the degradation products do not retain any toxic property. Finally, the mass spectrometry analysis of degradation products showed that the SF polypeptides recovered in the incubation buffers were representative of the aminoacidic sequence of the fibroin light chain and of the highly repetitive fibroin heavy chain, indicating that virtually the entire sequence of the fibroin protein constituent of SilkBridge was degraded.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

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), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-ß-Sheet (PIRIBS) [2] or ß-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 ß-solenoid (4RßS) [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 4RßS 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 ß-solenoid. Importantly, the 4RßS arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. This study provides 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.

Ok Google, how could I design therapeutics against prion diseases?

A number of previous successful attempts in the search for therapeutics for a variety of human pathologies highlight the importance of computational technologies in the drug discovery pipeline. This approach, often referred to as computer-aided drug design, is unfortunately inapplicable when the precise information regarding the three-dimensional structure of disease-associated proteins or the mechanism by which they are altered to generate misfolded isoforms are missing. A typical example is represented by prion diseases, fatal pathologies of the nervous system characterized by the conformational conversion of a physiological protein called PrPC into a misfolded and infectious isoform referred to as PrPSc. Missing information regarding the atomic structure of PrPSc as well as the mechanism of templated conversion of PrPC has severely halted the discovery of effective therapies for prion diseases. In this manuscript, we review emerging opportunities to apply computer-aided techniques to target PrPC, PrPSc or to design inhibitors of prion replication, and discuss how these fast-evolving technologies could lay the groundwork for the application of entirely novel rational drug design schemes for these devastating pathologies.