Biology subjects

Saibil, H. R.

Publications and source records attributed to Saibil, H. R..

4 recordsLinked to original sources

bioRxiv2021

2.7 A cryo-EM structure of ex vivo RML prion fibrils

Mammalian prions are lethal infectious agents that propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP), often referred to as prion rods. The structural features that define infectious prion rods and the molecular determinants of prion strain diversity are poorly understood. Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94-225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP primary sequence and/or represent distinguishing features of the distinct prion strains. These conformational changes impact the overall geometry of the fibrils and may also impact fibril pairing, one or both of which may critically influence PrP glycoform selection that occurs during strain-specific prion propagation.

biochemistry↗

bioRxiv2021

Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of -synuclein, huntingtin or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Recruitment and clustering of Hsc70 on the fibrils appear to be necessary for disassembly. Here we use atomic force microscopy (AFM) to show that segments of in vitro assembled -synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography reveals fibrils with regions of densely bound chaperones extending from the fibre surface, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of [~]4 subunits per second.

biochemistry↗

bioRxiv2021

The pore conformation of lymphocyte perforin

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with pro-apoptotic granzymes, to bind to the plasma membrane of the target cell where it oligomerises and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here we present a 4 [A] resolution cryo-EM structure of the perforin pore, revealing new inter- and intra-molecular interactions stabilising the pore. During assembly and pore formation, the helix-turn-helix motif at the bend in the central {beta}-sheet moves away from the bend to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerisation and membrane insertion, and explain how several pathogenic mutations affect function.

biochemistry↗

bioRxiv2021

Correlative light and electron microscopy reveals that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease

Huntingtons disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (i) multivesicular bodies (MVBs), or amphisomes and (ii) autolysosomes or residual bodies. The organelles were often found in close proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

neuroscience↗