Abstract Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases 1 . However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial cellular functions 2 . Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood. Here we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that PK forms stress-dependent reversible amyloids through a pH-sensitive amyloid core. Stress- induced cytosolic acidification promotes aggregate formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Our work thus unravels a conserved and potentially widespread mechanism underlying amyloid functionality and reversibility, fine-tuned to the respective physiological cellular pH range.
U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon-intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.
Raw data for the single particle analysis of GPI-anchorless PrP 27-30 amyloid fibrils.This is the data set for the analysis that uses a reference for the alignment and classification process. The files can be read with the image processing packages EMAN, IMAGIC, and RELION.
Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy ("cryo-EM"), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any "a priori" normalization routinely applied to the raw image data during collection ("flat field correction"). Our straightforward "a posteriori" correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over all spatial frequencies. The image sensor characteristics can also be measured continuously and used for correcting upcoming images.
The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.
The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.
Abstract Zelluläres Leben erfordert ein hohes Maß an molekularer Komplexität und Selbstorganisation, von denen einige in einem präbiotischen Kontext entstanden sein müssen. Hier demonstrieren wir, wie beide dieser Eigenschaften in einem plausibel präbiotischen System entstehen können. Wir fanden heraus, dass chemische Gradienten in einfachen Mischungen von aktivierten Aminosäuren und Fettsäuren zur Bildung von amyloidartigen Peptidfibrillen führen können, die innerhalb eines protozellulären Kompartiments lokalisiert sind. Bei diesem Prozess wirken die Fettsäure‐ oder Lipidvesikel sowohl als Filter, der die selektive Passage von aktivierten Aminosäuren ermöglicht, als auch als Barriere, welche die Diffusion der amyloidogenen Peptide blockiert, die sich spontan innerhalb der Vesikel bilden. Diese Synergie zwischen zwei unterschiedlichen Bausteinen des Lebens induziert eine signifikante Zunahme der molekularen Komplexität und räumlichen Ordnung und bietet damit einen Weg für die frühe molekulare Evolution, die zu einer lebenden Zelle führen könnte.
Bacterial contractile injection systems (CIS) are phage tail-like macromolecular complexes that mediate cell-cell interactions by injecting effector proteins into target cells. CIS from Streptomyces coelicolor (CIS Sc ) are localized in the cytoplasm. Under stress, they induce cell death and impact the bacteria’s life cycle. It remains unknown whether CIS Sc require accessory proteins to directly interact with the cytoplasmic membrane and function.Here, we characterize the putative membrane adaptor CisA, a conserved factor in CIS gene clusters across Streptomyces species. We show by cryo-electron tomography imaging and in vivo assays that CIS Sc contraction and function depend on CisA. Using single-particle cryo-electron microscopy, we provide an atomic model of the extended CIS Sc apparatus; however, CisA is not part of the complex. Instead, our findings show that CisA is a membrane protein with a cytoplasmic N-terminus predicted to interact with CIS Sc components, thereby providing a possible mechanism for mediating CIS Sc recruitment to the membrane and subsequent firing.Our work shows that CIS function in multicellular bacteria is distinct from Type 6 Secretion Systems and extracellular CIS, and possibly evolved due to the role CIS Sc play in regulated cell death.
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