Biology subjects

Picotti, P.

Publications and source records attributed to Picotti, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Chemically induced USP21-KLHL12 association recruits USP21 to COPII assembly

Ubiquitin-specific protease 21 (USP21) is a deubiquitinase implicated in various biological processes and disease states, including cancer. To better understand USP21 protein interaction networks, we used biotin identification proximity labeling, and uncovered USP21 interactions with the cytoskeleton, endosomal, and transcriptional regulators. Notably, a subset of these interactions was modulated by the USP21 selective inhibitor BAY-805. In cells, BAY-805 promoted proximity between USP21 and Kelch like protein KLHL12, a component of the Cullin3 ligase complex. Structural modeling using AlphaFold3, together with hydrogen deuterium exchange mass spectrometry, identified an orthosteric BAY-805 binding site within USP21. Binding of BAY-805 functionally competes with ubiquitin at USP21, thereby enabling USP21 interaction with KLHL12. This BAY-805 induced proximity elicited relocalization of USP21 to KLHL12 containing coat protein complex II (COPII) vesicles. Induced USP21-KLHL12 association led to enlargement of COPII vesicles and impaired transport of large cargoes such as collagen. Overall, our findings reveal a novel chemically induced proximity mechanism in which the monovalent ligand BAY-805 outcompetes ubiquitin from USP21, thereby recruiting USP21 to KLHL12 associated protein complexes.

cell biology↗

bioRxiv2026

The Human Bindome: A Proteome-scale Atlas of Designed Binder Candidates

Affinity reagents such as antibodies are indispensable for interrogating proteins biological function. Yet they are costly and frequently unreliable, with unknown sequences, posing challenges to reproducible experimental research. Deep learning-based protein design can now in silico generate affinity reagents achieving reliable experimental success rates, but has remained largely confined to specialist laboratories. Here we present the Human Bindome, a proteome-scale atlas of high-confidence in silico protein binder candidates. By embedding the experimentally benchmarked BindCraft method in an accelerated, parallelized framework with automated domain-level target selection, we generated 306,146 binder candidates covering 8,296 human proteins (40.9% of the full proteome). Every candidate carries a defined sequence, a predicted binder-target structure model, and in silico confidence metrics. We characterize proteome-wide coverage and show that binder epitopes frequently overlap functional sites. This positions the Bindome as a resource of genetically encodable perturbagens for site-specific, modular control of protein function. The Bindome is freely available through a web interface (https://bindome.epfl.ch), with agentic, natural-language querying and as data splits for machine-learning model development. We anticipate that the Bindome will be valuable for the scientific community by providing affinity and perturbation reagents with broad applications in dissecting biological mechanisms as well as in drug and target discovery.

synthetic biology↗

bioRxiv2026

Weak interactions drive selective proteome demixing and tune the differential response to environmental perturbations

The intracellular space is a crowded environment where macromolecules perform distinct tasks despite pervasive "non-specific" interactions. Whether these interactions are functionally relevant and how they influence cellular organization remains unclear. Here, we developed QuPID-MS, which measures the propensity of proteins to phase separate in native cell extracts proteome-wide. We find that weak interactions drive condensation of half of the proteome in a crowding- and temperature-dependent manner and we present evidence that this proteome demixing occurs in cells. Importantly, protein condensation properties are conserved and broadly change when cells adapt to new environments, demonstrating that weak interactions are regulated and linked to function. Indeed, condensation of the growth regulator TORC1 coincides with its rapid inactivation, while high solubility of the stress-activated Hog1 ensures its activity across conditions. We thus uncover a fundamental organizing principle that allows tuning of cell growth to environmental fluctuations while ensuring other processes function robustly despite perturbations.

cell biology↗

bioRxiv2026

Deconvolving the structural heterogeneity of alpha-Synuclein in vitro and in situ

The structural states of proteins in cells and tissues provide important insight into their functional states, but studying protein structures in situ remains challenging. Furthermore, a single protein can adopt multiple conformations in cells, which typically cannot be assessed by most structural approaches. Here we developed a novel approach, based on structural proteomics fingerprints, for the quantitative analysis of the distribution of structural states of a protein in vitro and in situ. We applied it to the Parkinsons disease hallmark protein alpha-synuclein (aSyn), for which various structural states (disordered, helical, oligomeric and amyloid fibrillar, among others) have been characterized in vitro, but for which the in vivo structural states remain hotly debated. We measured structure-specific proteolytic fingerprints from well-characterized aSyn in vitro conformations and used them to quantitatively determine the aSyn conformational composition in samples of interest. We first benchmarked our approach using ground truth datasets of known composition and showed that, during in vitro amyloid fibril formation, we could simultaneously detect a time-dependent decrease in disordered monomeric aSyn, an increase in {beta}-sheet-rich oligomers, and a delayed rise in amyloid fibrils. We then applied the method to complex, biologically relevant samples. In a S. cerevisiae aSyn overex-pression model, aSyn was predominantly helical, with an increased helical fraction accompanying its relocalization from the plasma membrane to cytosolic lipid droplets. This shift was linked to proteome-wide changes in lipid droplet homeostasis and fatty acid and ergosterol metabolism, underscoring the role of lipid metabolism and droplet formation in aSyn biology. Importantly, we also detected helical aSyn in human iPSC-derived cortical neurons, supporting the physiological relevance of this conformation. Finally, neurons differentiated from PD patient-derived iPSCs showed elevated levels of {beta}-sheet-rich aSyn compared to wild-type cells. Our approach allowed the in situ identification and quantification of different structural states of aSyn directly in cell lysates. Since several proteins can adopt multiple, functionally-relevant conformations in cells, our approach should be broadly applicable to in situ, quantitative structural and functional studies of proteins.

neuroscience↗

bioRxiv2026

Neuronal calcium sensors emerge as novel adenylyl cyclase 8 regulators

Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal calcium sensor hippocalcin-like protein 1 (HPCAL1) as a direct interaction partner of AC8. Using biochemistry, structural proteomics, integrative modeling, cryo-EM and cell-based FRET approaches, we show that HPCAL1 associates with AC8 in a Ca2+-dependent manner. HPCAL1 shows weak positive modulation of AC8 activity in vitro, with sub micromolar affinity. HPCAL1 interacts with flexible regulatory regions of AC8, including the C1b domain and the N- and C-terminal regions. Interestingly, other members of the neuronal calcium sensor family can bind AC8 via the same sites. Together, our study reveals a previously unrecognized mode of Ca2+-dependent AC8 recognition by a group of neuronal calcium sensor proteins, which may be relevant to context-dependent regulation of cAMP signaling.

biochemistry↗

bioRxiv2026

In-source fragmentation in mass spectrometry-based proteomics: prevalence, impact, and strategies for mitigation

Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide mixtures and the first mass filter of a tandem mass spectrometer (MS). To understand the prevalence and properties of ISF, we generated 13 proteomics datasets and analyzed them along with additional 25 previously published datasets spanning a broad range of sample types, MS, and proteomics approaches including classical bottom-up proteomics, immunopeptidomics, structural proteomics, and phosphoproteomics. We found that, in typical trypsin-digested samples on average 1 % of fully-tryptic peptides and 22 % of semi-tryptic peptides originated from ISF. However, we observed large variations between datasets, and in-source fragments exceeded, in some cases, a third of the total peptide identifications. The extent of ISF was dependent on the peptide sequence, the instrument, method parameters, and sample complexity. Although ISF did not impair relative quantification across samples, it generated peptides that could be misinterpreted qualitatively, inflated peptide identifications, and comprised up to 37 percent of peptides shorter than 9 amino acids in immunopeptidomics datasets. We propose that, for peptide-centric applications, our open-source ISF detection approach be used to re-annotate peptides generated by ISF and remove them to avoid misinterpretation of data. ISF is an increasing concern with improving mass spectrometers, as they enable detection of an ever-increasing number of m/z features, including low abundance features like ISF products. Our work thus addresses a growing issue in proteomics and presents solutions to mitigate the impact of in-source fragment peptides. In the future, improved feature detection algorithms may enable elucidation of new ISF patterns affecting side chains that have been missed so far, which could contribute to explaining the vast space of as-yet unannotated proteomics data.

biochemistry↗

bioRxiv2025

Systematic structural and functional analysis of metal-binding sites in native proteomes

Metal-binding proteins (MBPs) are widespread and critical for many biological processes. Proteomics-based approaches have identified MBPs in various systems but typically cannot determine the location of metal-binding sites or shed light on the functional roles of metals. Thus, a systems-wide map of metal binding sites and an understanding of the consequences of metal binding for protein function are still lacking. Here, we used limited proteolysis-coupled mass spectrometry (LiP-MS) combined with chelator treatment to globally map metal-protein interactions and binding sites in native protein lysates. We applied the pipeline, named Metal-LiP, to Escherichia coli, identified 981 peptides on 297 proteins that exhibited dose responsiveness to chelator treatment, and quantified the relative metal-binding affinity. These proteins are strongly enriched for known or predicted MBPs, based on our novel compiled ground truth dataset of previously annotated (n=1013) and predicted (n=850) MBPs. Further, chelator-responsive peptides mapped at or near annotated metal-binding sites in 3D protein structures, showing that Metal-LiP can identify metal-binding sites, and provided novel experimental evidence for previously predicted sites. Notably, Metal-LiP identified 225 novel candidate MBPs and we validated five of these novel MBPs (GatZ, AhpC, GrxB, MsrC, TrhO) using inductively coupled plasma-mass spectrometry to detect metal binding in purified proteins. Finally, we used orthogonal approaches to probe the broad functional consequences of metal binding. Using a combination of Metal-LiP, thermal proteome profiling, size-exclusion chromatography coupled to mass spectrometry, and structural analyses, we identified cases where metal binding affects protein stability, modulates the assembly states of protein complexes, or is likely to affect enzymatic activity. We thereby propose functional roles for metal binding in 98 of our Metal-LiP hits and in 225 proteins in total. In summary, we describe an approach to identify bona fide MBPs with binding site-level resolution across the proteome. We have combined it with other methods to identify 424 candidate MBPs in the E coli proteome, to map known and novel candidate metal-binding sites for 297 of these proteins, and to suggest functional roles of metal binding.

systems biology↗

bioRxiv2025

Allosteric Gain-of-Function and Off-Mechanism Effects of ATP-Competitive Kinase Inhibitors Revealed by Multimodal Proteomics

ATP competitive kinase inhibitors represent one of the largest classes of targeted anti-cancer drugs. While their primary mechanism is to block catalytic activity, they can also trigger paradoxical phenotypic effects that cannot be explained by catalytic inhibition alone. These observations point to a hidden layer of drug action that modulates non-catalytic kinase functions via changes in kinase conformation and protein-protein interactions (PPIs). Here we developed a multimodal proteomics approach combining limited proteolysis coupled mass spectrometry on affinity purified samples (AP-LiP-MS), AP-MS, and proximity labeling-MS to map inhibitor-induced conformation and PPI changes. We show that inhibitor binding causes structural rearrangements in the AIDs of all tested kinases, consistent with a transition to an open, active-like kinase conformation. These structural shifts drive distinct kinase-protein interaction changes that control non-catalytic functions: sequestration of AMPK by inhibited CAMKK2 blocks phosphorylation by other kinases, CHEK1 inhibition causes dissociation from the mitochondrial protein CLPB and leads to mitochondrial fragmentation, and structural changes in inhibited PRKCA trigger rapid relocalization to cell junctions. Thus we identify the ATP-binding site as a major organizing center of kinase conformation and interaction. Our work suggests that these on-target, off-mechanism effects are highly prevalent and provides the analytical framework to systematically characterize a frequently overlooked phenomenon highly relevant for understanding drug side effects to guide the development of novel therapeutics.

molecular biology↗

bioRxiv2025

Structure-function relationship of alpha-synuclein fibrillar polymorphs derived from distinct synucleinopathies

The aggregation of the protein alpha-synuclein (Syn) is a common feature of multiple neurodegenerative diseases collectively called synucleinopathies, for which the pathobiology is not well understood. The different phenotypic characteristics of the synucleinopathies Parkinsons disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) have been proposed to originate from the distinct structures adopted by Syn in its amyloid forms. Here, using covalent labeling and limited proteolysis coupled to mass spectrometry (LiP-MS) in vitro and in situ within neuronal cells and directly in native patient brain homogenates, we show that pathogenic Syn from distinct synucleinopathies (PD, DLB and MSA) are structurally different. Further, we found that fibrillar structural differences are associated with different fibril interactomes and neuronal responses. We discovered disease-specific ubiquitination patterns and turnover profiles for pathogenic Syn species, detected molecular pathways responding specifically to the uptake of different Syn fibrillar polymorphs, and identified a subset of the involved proteins as candidate direct interactors of Syn. In particular, components of the Ubiquitin-proteasomal System (UPS), including E3 ubiquitin ligases, chaperones, and Deubiquitinating proteins, showed disease/polymorph-specific interaction patterns, possibly accounting for different resistance of patient-derived Syn fibrils to degradation. Genetic modulation with CRISPR-based tools showed that members of the UPS degradation pathway (three E3 ligases: UBE3A, TRIM25, HUWE1 and the AAA+ ATPase VCP) reduced Syn inclusions, in a strain-specific manner. LiP-MS also identified sets of proteins with altered protease susceptibility in postmortem brain homogenates of PD, DLB, and MSA patients. These sets were largely disease-specific and included proteins altered in cells treated with fibrils derived from patients with the matching disease. Our findings provide insight into cellular processes involved in the accumulation and turnover of Syn pathogenic aggregates in PD, DLB and MSA in a disease specific manner and constitutes a resource of potential novel drug targets in these synucleinopathies.

systems biology↗

bioRxiv2025

The environmental stress response controls the biophysical properties of the cytoplasm and is critical for survival in quiescence

All organisms have evolved survival strategies to cope with changes in environmental conditions. Nutrient deprivation, one of the most frequently encountered stresses in nature, causes haploid budding yeast to enter a reversible state of non-proliferation known as quiescence, which entails extensive remodeling of gene expression, metabolism and the cellular biophysical properties. Yeast cells can adapt to and survive long periods of time in glucose starvation-induced quiescence, provided they are able to respire in the early stages of glucose withdrawal. When respiration is blocked during glucose withdrawal, cells prematurely age and exhibit markedly reduced survival and cytoplasmic diffusion. We find here that respiration is required to induce a quiescence-related gene expression program. Induction of this program prior to withdrawing glucose in respiration-inhibited cells bypasses the need for respiration and rescues survival and biophysical properties to levels seen in glucose-starved but respiration-competent cells. This rescue effect relies on proteomic adaptation, which partially occurs through inactivation of Ras/PKA signaling and activation of the environmental stress response via the transcription factors Msn2/4. This signaling cascade triggers the expression of stress response genes and modulates the cytoplasmic diffusion state of cells, ensuring long-term survival in quiescence even in the absence of respiration. Our results highlight the importance of stress adaptation in quiescence and aging, integrating gene expression control and modulation of cytoplasmic properties to maintain cell fitness.

cell biology↗

bioRxiv2025

Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

The function of a protein is determined by its structure, which may change dynamically in response to post-translational modifications, interaction with other molecules, or environmental factors like temperature. Limited proteolysis-coupled mass spectrometry (LiP-MS) captures such structural alterations on a proteome-wide scale via the detection of altered protease susceptibility patterns of proteins. However, this technique has so far required cell lysis, which exposes proteins to non-native conditions and can disrupt labile interactions such as those occurring within biomolecular condensates. To study protein structures directly within cells, we developed in-cell LiP-MS. We optimized conditions for introduction of proteinase K into human cells using electroporation and validated that intracellular cleavage occurs. In-cell LiP-MS captured not only the known binding of rapamycin to FKBP1A within the cell, but also downstream effects of pathway activation. Moreover, it detected global protein structural alterations upon sodium arsenite treatment and captured the structural dynamics of hundreds of proteins from biomolecular condensates with peptide level resolution and within live human cells. We detected known and novel structural alterations of proteins from stress granules as well as from nuclear speckles and validated alteration of nuclear speckles by fluorescence microscopy. Our dataset provides a resource describing the structural changes of human proteins in response to cellular stress and pinpoints structurally altered regions. Further, comparison of LiP-based structural fingerprints before and after cell lysis revealed which human proteins are susceptible to structural change upon cell lysis, therefore guiding the design of future experiments requiring native protein structures.

systems biology↗

bioRxiv2024

The Main protease (Mpro) from SARS-CoV-2 triggers plasma clotting in vitro by activating coagulation factors VII and FXII

Although the connection between COVID-19 and coagulopathy has been clear since the early days of SARS-CoV-2 pandemic, the underlying molecular mechanisms remain unclear. Available data support that the burst of cytokines and bradykinin, observed in some COVID-19 patients, sustains systemic inflammation and the hypercoagulant state, thus increasing thrombotic risk. Here we show that the SARS-CoV-2 main protease (Mpro) can play a direct role in the activation of the coagulation cascade. Adding Mpro to human plasma from healthy donors increased clotting probability by 2.5-fold. The results of enzymatic assays and degradomics analysis indicate that Mpro triggers plasma clotting by proteolytically activating coagulation factors zymogens VII and XII at their physiological activation sites, i.e. Arg152-Ile153 bond for FVII and Arg353-Val354 bond for FXII, where FVII and FXII are strategically positioned at the very beginning of the extrinsic or intrinsic pathways of blood coagulation. These findings are not compatible with the substrate specificity of the protease known so far, displaying a prevalence for a Gln-residue in P1 and a hydrophobic amino acid in P2 position. This apparent discrepancy was resolved by High Throughput Protease Screen assay, unveiling an extended, time-dependent, secondary specificity of Mpro for Arg-X bonds, which was further confirmed by Hydrogen-Deuterium Exchange Mass spectrometry analysis of Arg-containing inhibitors binding to Mpro and by enzymatic assays showing that the protease can cleave peptide substrates containing Arg in P1. Overall, integrating biochemical, proteomics and structural biology experiments, we unveil a novel mechanism linking SARS-CoV-2 infection to thrombotic complications in COVID-19.

biochemistry↗

bioRxiv2024

Enhancing the biological insight from limited proteolysis-coupled mass spectrometry

Limited proteolysis combined with mass spectrometry (LiP-MS) facilitates probing structural changes on a proteome-wide scale in situ. Distinguishing the different signal contributions, such as changes in protein abundance, from protein abundance changes remains challenging. We propose a two-step approach, first removing unwanted variations from the LiP signal that are not caused by protein structural effects and subsequently inferring the effects of variables of interest on the remaining signal. Using LiP-MS data from three species we demonstrate that our framework provides a uniquely powerful approach for deconvolving LiP-MS signals and inferring protein structural changes.

bioinformatics↗

bioRxiv2023

An approach to characterize mechanisms of action of anti-amyloidogenic compounds in vitro and in situ

Aggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probed in vitro using purified protein samples, even though these models may not capture in vivo protein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinsons disease (PD) protein -Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of -Synuclein in complex cell and tissue extracts and identify off-targets. In vitro analyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured -Synuclein monomer, detected preferential interactions of ThT with the N-terminus of -Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with -Synuclein monomers. Importantly, in mammalian cell lysates, -Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compounds in vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. These in situ analyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than -Synuclein and showing that anti-amyloidogenic compounds should be analyzed in situ as well as in vitro. Our modular pipeline will enable in situ screening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound action in vitro.

systems biology↗

bioRxiv2023

Structural basis of connexin-36 gap junction channel inhibition

Connexin gap junction channels and hemichannels play important roles in intercellular communication and signaling. Some of connexin isoforms are associated with diseases, including hereditary neuropathies, heart disease and cancer. Although small molecule inhibitors of connexins show promise as therapeutic agents, the molecular mechanisms of connexin channel inhibition are unknown. Here, we report the cryo-EM structure of connexin-36 (Cx36) bound to an anti-malarial drug mefloquine at 2.1 [A] resolution. Six drug binding sites partially occlude the pore of each connexon forming the channel. Each drug molecule in the ring makes contacts with residues in the pore-lining pocket and with the neighbouring mefloquine molecules, partially occluding the pore and modifying the pore electrostatics, ultimately reducing solute translocation through the channel. Structures of Cx36 in the presence of quinine and quinidine show a similar mode of drug binding. Molecular dynamics simulations of Cx36 bound to mefloquine show that drug binding affects the kinetics of ion passage through the pore. This previously undescribed mode of connexin channel inhibition presents an opportunity for designing subtype-specific connexin inhibitors. One-sentence summaryMechanism of connexin channel inhibition by small molecules

biophysics↗

bioRxiv2023

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seeding experiments

The aggregation of the protein -synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the -synuclein fibrils hampers efforts in disease-relevant in vitro studies on -synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of -synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4 a pH- dependent selection between Types 1, 2 and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

biophysics↗

bioRxiv2023

Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels

In myelinating Schwann cells, communication between myelin layers is mediated by gap junction channels (GJC) formed by docked connexin 32 hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy with no cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high resolution cryo-EM structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: in the W3S and R22G mutant HCs, the N-terminal helix partially occludes the pore, consistent with an impaired HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X. One-Sentence SummaryConnexin 32 channel structures reveal a gating helix defect in CMT1X disease-associated mutant hemichannels

biophysics↗

bioRxiv2023

Structural basis of activation and inhibition of the Ca2+/calmodulin-sensitive adenylyl cyclase 8

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, including cognitive functions and memory, the structural basis of its regulation by G proteins and CaM is not well defined. Here we report the 3.5 [A] resolution cryo-EM structure of the bovine AC8 bound to Ca2+/CaM and the stimulatory Gs protein. The structure reveals the architecture of the ordered AC8 domains bound to Gs and a small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry, allow us to identify the contact sites between AC8 and its regulators, CaM, Gs, and G{beta}{gamma}, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

biochemistry↗