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Nature Medicine Jun 2023Abnormal α-synuclein aggregation is a key pathological feature of a group of neurodegenerative diseases known as synucleinopathies, which include Parkinson's disease...
Abnormal α-synuclein aggregation is a key pathological feature of a group of neurodegenerative diseases known as synucleinopathies, which include Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy (MSA). The pathogenic β-sheet seed conformation of α-synuclein is found in various tissues, suggesting potential as a biomarker, but few studies have been able to reliably detect these seeds in serum samples. In this study, we developed a modified assay system, called immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC), which enables the detection of pathogenic α-synuclein seeds in the serum of individuals with synucleinopathies. In our internal first and second cohorts, IP/RT-QuIC showed high diagnostic performance for differentiating PD versus controls (area under the curve (AUC): 0.96 (95% confidence interval (CI) 0.95-0.99)/AUC: 0.93 (95% CI 0.84-1.00)) and MSA versus controls (AUC: 0.64 (95% CI 0.49-0.79)/AUC: 0.73 (95% CI 0.49-0.98)). IP/RT-QuIC also showed high diagnostic performance in differentiating individuals with PD (AUC: 0.86 (95% CI 0.74-0.99)) and MSA (AUC: 0.80 (95% CI 0.65-0.97)) from controls in a blinded external cohort. Notably, amplified seeds maintained disease-specific properties, allowing the differentiation of samples from individuals with PD versus MSA. In summary, here we present a novel platform that may allow the detection of individuals with synucleinopathies using serum samples.
Topics: Humans; alpha-Synuclein; Synucleinopathies; Parkinson Disease; Multiple System Atrophy; Biomarkers; Lewy Body Disease
PubMed: 37248302
DOI: 10.1038/s41591-023-02358-9 -
Nature Feb 2024Thousands of proteins have been validated genetically as therapeutic targets for human diseases. However, very few have been successfully targeted, and many are...
Thousands of proteins have been validated genetically as therapeutic targets for human diseases. However, very few have been successfully targeted, and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions-direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central β-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.
Topics: Humans; Allosteric Regulation; Allosteric Site; Mutation; Protein Binding; Protein Folding; Proto-Oncogene Proteins p21(ras); Reproducibility of Results; Substrate Specificity; Thermodynamics
PubMed: 38109937
DOI: 10.1038/s41586-023-06954-0 -
Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.Science (New York, N.Y.) Jan 2024β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven...
β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues.
Topics: beta-Arrestins; Cryoelectron Microscopy; Receptors, G-Protein-Coupled; Signal Transduction; Protein Conformation, beta-Strand; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Humans
PubMed: 38175886
DOI: 10.1126/science.adj3347 -
Science Advances Sep 2023Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is...
Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and forming cross-β fibers. Fibrillation complicates insulin storage and therapeutic application. Molecular details of insulin fibrillation remain unclear, hindering efforts to prevent fibrillation process. Here, we characterized insulin fibrils using cryo-electron microscopy (cryo-EM), showing multiple forms that contain one or more of the protofilaments containing both the A and B chains of insulin linked by disulfide bonds. We solved the cryo-EM structure of one of the fibril forms composed of two protofilaments at 3.2-Å resolution, which reveals both the β sheet conformation of the protofilament and the packing interaction between them that underlie the fibrillation. On the basis of this structure, we designed several insulin mutants that display reduced fibrillation while maintaining native IR signaling activity. These designed insulin analogs may be developed into more effective therapeutics for type 1 diabetes.
Topics: Humans; Cryoelectron Microscopy; Diabetes Mellitus, Type 1; Insulin; Protein Aggregates
PubMed: 37713485
DOI: 10.1126/sciadv.adi1057 -
Nature Jan 2024Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for...
Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, β-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.
Topics: Humans; Alzheimer Disease; Amyloid; Chronic Traumatic Encephalopathy; Cryoelectron Microscopy; Neurofibrillary Tangles; tau Proteins; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Time Factors
PubMed: 38030728
DOI: 10.1038/s41586-023-06788-w -
Cells Dec 2023Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned...
Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, and (Tasar) silk microfibres were embedded within self-assembling silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, and Tasar microfibres were prepared with lengths between 250 and 500 μm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (/) concentrations, into 3% (/) silk solutions during the solution-gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (/) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (/) > 10% (/) > unmodified hydrogels for either fibre type, although fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential 'flow points' within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (/) microfibres as well as 2% (/) and 10% (/) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres.
Topics: Humans; Animals; Silk; Induced Pluripotent Stem Cells; Bombyx; Cell-Matrix Junctions; Hydrogels
PubMed: 38201214
DOI: 10.3390/cells13010010 -
Allergologie Select 2023Hymenoptera venom (HV) is injected into the skin during a sting by Hymenoptera such as bees or wasps. Some components of HV are potential allergens and can cause large...
Diagnosis and treatment of Hymenoptera venom allergy: S2k Guideline of the German Society of Allergology and Clinical Immunology (DGAKI) in collaboration with the Arbeitsgemeinschaft für Berufs- und Umweltdermatologie e.V. (ABD), the Medical Association of German Allergologists (AeDA), the German...
Hymenoptera venom (HV) is injected into the skin during a sting by Hymenoptera such as bees or wasps. Some components of HV are potential allergens and can cause large local and/or systemic allergic reactions (SAR) in sensitized individuals. During their lifetime, ~ 3% of the general population will develop SAR following a Hymenoptera sting. This guideline presents the diagnostic and therapeutic approach to SAR following Hymenoptera stings. Symptomatic therapy is usually required after a severe local reaction, but specific diagnosis or allergen immunotherapy (AIT) with HV (VIT) is not necessary. When taking a patient's medical history after SAR, clinicians should discuss possible risk factors for more frequent stings and more severe anaphylactic reactions. The most important risk factors for more severe SAR are mast cell disease and, especially in children, uncontrolled asthma. Therefore, if the SAR extends beyond the skin (according to the Ring and Messmer classification: grade > I), the baseline serum tryptase concentration shall be measured and the skin shall be examined for possible mastocytosis. The medical history should also include questions specific to asthma symptoms. To demonstrate sensitization to HV, allergists shall determine concentrations of specific IgE antibodies (sIgE) to bee and/or vespid venoms, their constituents and other venoms as appropriate. If the results are negative less than 2 weeks after the sting, the tests shall be repeated (at least 4 - 6 weeks after the sting). If only sIgE to the total venom extracts have been determined, if there is double sensitization, or if the results are implausible, allergists shall determine sIgE to the different venom components. Skin testing may be omitted if in-vitro methods have provided a definitive diagnosis. If neither laboratory diagnosis nor skin testing has led to conclusive results, additional cellular testing can be performed. Therapy for HV allergy includes prophylaxis of reexposure, patient self treatment measures (including use of rescue medication) in the event of re-stings, and VIT. Following a grade I SAR and in the absence of other risk factors for repeated sting exposure or more severe anaphylaxis, it is not necessary to prescribe an adrenaline auto-injector (AAI) or to administer VIT. Under certain conditions, VIT can be administered even in the presence of previous grade I anaphylaxis, e.g., if there are additional risk factors or if quality of life would be reduced without VIT. Physicians should be aware of the contraindications to VIT, although they can be overridden in justified individual cases after weighing benefits and risks. The use of β-blockers and ACE inhibitors is not a contraindication to VIT. Patients should be informed about possible interactions. For VIT, the venom extract shall be used that, according to the patient's history and the results of the allergy diagnostics, was the trigger of the disease. If, in the case of double sensitization and an unclear history regarding the trigger, it is not possible to determine the culprit venom even with additional diagnostic procedures, VIT shall be performed with both venom extracts. The standard maintenance dose of VIT is 100 µg HV. In adult patients with bee venom allergy and an increased risk of sting exposure or particularly severe anaphylaxis, a maintenance dose of 200 µg can be considered from the start of VIT. Administration of a non-sedating H1-blocking antihistamine can be considered to reduce side effects. The maintenance dose should be given at 4-weekly intervals during the first year and, following the manufacturer's instructions, every 5 - 6 weeks from the second year, depending on the preparation used; if a depot preparation is used, the interval can be extended to 8 weeks from the third year onwards. If significant recurrent systemic reactions occur during VIT, clinicians shall identify and as possible eliminate co-factors that promote these reactions. If this is not possible or if there are no such co-factors, if prophylactic administration of an H1-blocking antihistamine is not effective, and if a higher dose of VIT has not led to tolerability of VIT, physicians should should consider additional treatment with an anti IgE antibody such as omalizumab as off lable use. For practical reasons, only a small number of patients are able to undergo sting challenge tests to check the success of the therapy, which requires in-hospital monitoring and emergency standby. To perform such a provocation test, patients must have tolerated VIT at the planned maintenance dose. In the event of treatment failure while on treatment with an ACE inhibitor, physicians should consider discontinuing the ACE inhibitor. In the absence of tolerance induction, physicians shall increase the maintenance dose (200 µg to a maximum of 400 µg in adults, maximum of 200 µg HV in children). If increasing the maintenance dose does not provide adequate protection and there are risk factors for a severe anaphylactic reaction, physicians should consider a co-medication based on an anti-IgE antibody (omalizumab; off-label use) during the insect flight season. In patients without specific risk factors, VIT can be discontinued after 3 - 5 years if maintenance therapy has been tolerated without recurrent anaphylactic events. Prolonged or permanent VIT can be considered in patients with mastocytosis, a history of cardiovascular or respiratory arrest due to Hymenoptera sting (severity grade IV), or other specific constellations associated with an increased individual risk of recurrent and/or severe SAR (e.g., hereditary α-tryptasemia). In cases of strongly increased, unavoidable insect exposure, adults may receive VIT until the end of intense contact. The prescription of an AAI can be omitted in patients with a history of SAR grade I and II when the maintenance dose of VIT has been reached and tolerated, provided that there are no additional risk factors. The same holds true once the VIT has been terminated after the regular treatment period. Patients with a history of SAR grade ≥ III reaction, or grade II reaction combined with additional factors that increase the risk of non response or repeated severe sting reactions, should carry an emergency kit, including an AAI, during VIT and after regular termination of the VIT.
PubMed: 37854067
DOI: 10.5414/ALX02430E -
Nature Neuroscience Jun 2023Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with...
Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a β-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.
Topics: Humans; Amyotrophic Lateral Sclerosis; TDP-43 Proteinopathies; Frontotemporal Lobar Degeneration; Frontotemporal Dementia; DNA-Binding Proteins
PubMed: 37248338
DOI: 10.1038/s41593-023-01341-4