Two-tier supramolecular encapsulation of small molecules in a protein cage

Expanding protein design to include other molecular building blocks has the potential to increase structural complexity and practical utility. Nature often employs hybrid systems, such as clathrin-coated vesicles, lipid droplets, and lipoproteins, which combine biopolymers and lipids to transport a broader range of cargo molecules. To recapitulate the structure and function of such composite compartments, we devised a supramolecular strategy that enables porous protein cages to encapsulate poorly water-soluble small molecule cargo through templated formation of a hydrophobic surfactant-based core. These lipoprotein-like complexes protect their cargo from sequestration by serum proteins and enhance the cellular uptake of fluorescent probes and cytotoxic drugs. This design concept could be applied to other protein cages, surfactant mixtures, and cargo molecules to generate unique hybrid architectures and functional capabilities.ISSN:2041-172

Protein Cages: From Fundamentals to Advanced Applications

Proteins that self-assemble into polyhedral shell-like structures are useful molecular containers both in nature and in the laboratory. Here we review efforts to repurpose diverse protein cages, including viral capsids, ferritins, bacterial microcompartments, and designed capsules, as vaccines, drug delivery vehicles, targeted imaging agents, nanoreactors, templates for controlled materials synthesis, building blocks for higher-order architectures, and more. A deep understanding of the principles underlying the construction, function, and evolution of natural systems has been key to tailoring selective cargo encapsulation and interactions with both biological systems and synthetic materials through protein engineering and directed evolution. The ability to adapt and design increasingly sophisticated capsid structures and functions stands to benefit the fields of catalysis, materials science, and medicine.ISSN:0009-2665ISSN:1520-689

Expanding the mass range for UVPD-based native top-down mass spectrometry

Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles

TAF15 amyloid filaments in frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy

Thermal stabilization of diverse biologics using reversible hydrogels

Improving the thermal stability of biologics, including vaccines, is critical to reduce the economic costs and health risks associated with the cold chain. Here, we designed a versatile, safe, and easy-to-use reversible PEG-based hydrogel platform formed via dynamic covalent boronic ester cross-linking for the encapsulation, stabilization, and on-demand release of biologics. Using these reversible hydrogels, we thermally stabilized a wide range of biologics up to 65°C, including model enzymes, heat-sensitive clinical diagnostic enzymes (DNA gyrase and topoisomerase I), protein-based vaccines (H5N1 hemagglutinin), and whole viruses (adenovirus type 5). Our data support a generalized protection mechanism for the thermal stabilization of diverse biologics using direct encapsulation in reversible hydrogels. Furthermore, preliminary toxicology data suggest that the components of our hydrogel are safe for in vivo use. Our reversible hydrogel platform offers a simple material solution to mitigate the costs and risks associated with reliance on a continuous cold chain for biologic transport and storageISSN:2375-254

TAF15 amyloid filaments in frontotemporal lobar degeneration.

Acknowledgements: We thank the individuals and their families for donating brain tissue; the Queen Square Brain Bank for Neurological Disorders at University College London Queen Square Institute of Neurology, which receives support from the Reta Lila Weston Institute for Neurological Studies, for supplying tissue from individuals 2 and 3; J. Grafman for supplying tissue from individual 4; M. Jacobsen for help with neuropathological examinations; R. Richardson, K. Cox and N. Maynard for help with histology and immunohistochemistry; J. Grimmett, T. Darling and I. Clayson for help with high-performance computing; K. Yamashita and G. Murshudov for help with model refinements; and T. Behr, A. Bertolotti, R. Chen, S. Davies, M. Goedert, D. Hilvert and S. Scheres for discussions. This work was supported by the electron microscopy and scientific computing facilities at the MRC Laboratory of Molecular Biology and by the Center for Medical Genomics at the Indiana University School of Medicine. This work was supported by the Medical Research Council as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) (no. MC_UP_1201/25 to B.R.-F.); the US National Institutes of Health (nos. U01-NS110437, RF1-AG071177 and R01-AG080001 to R.V. and B.G.); the Alzheimer’s Society (nos. AS-PG-18-004 and AS-PG-21-004 to T.L.); the Association for Frontotemporal Degeneration (no. 2019-0009 to Y.B. and T.L.); a Swiss National Science Foundation Postdoctoral Fellowship (no. P500PB_206890 to S.T.); and a Leverhulme Early Career Fellowship (no. ECF-2022-610 to D.A.). For the purposes of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising.Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy

Tau filaments from amyotrophic lateral sclerosis/parkinsonism-dementia complex adopt the CTE fold

Peer reviewed: TruePublication status: PublishedThe amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors.</jats:p

Tau filaments from amyotrophic lateral sclerosis/parkinsonism-dementia complex adopt the CTE fold.

Peer reviewed: TruePublication status: PublishedThe amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors