Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy/tomography on post-mortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many, but not all aSyn inclusions. Crowding of organellar components was confirmed by STED-based superresolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIRimaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the PD brain.

No abstract

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

No abstract

No abstract

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in ␤-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric ␤-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into ␤-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric ␤-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed ␤-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is ؎1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils. T he ability of soluble proteins or protein fragments to convert spontaneously into amyloid fibrils under some circumstances is a very important biological phenomenon, because this process is related to a range of human disorders such as spongiform encephalopathies, Alzheimer's disease, etc. (1, 2). Although soluble precursors of amyloidogenic proteins do not have any obvious sequence homology or common folding patterns, x-ray fiber diffraction data indicate that all amyloid fibrils share a characteristic cross-␤-structure (3, 4). This finding suggests that the key elements of the fibril formation process may be common to all proteins and that, therefore, a highly simplified system that is able to polymerize into ␤-sheets can offer additional insights into the molecular details of amyloid fibril formation. Up till now, the search for such simple model systems has been based mainly on empirical approaches such as, for example, the identification of fragments of amyloidogenic proteins that can self-assemble into amyloid fibrils (5-8). Some rational designs based on alternating hydrophobic and hydrophilic residues have been found to have some success in identifying the interactions behind ␤-sheet polymerization (9). Although such systems have helped to clarify many aspects of this process, our detailed understanding of the factors promoting amyloid formation remains limited.To probe the interactions driving ␤-sheet aggregation, we have investigated the effect of specific residues on the propensity of a given sequence to form amyloid fibrils. To this end, we have used a computer-based method (10-12) to design a series of self-associating hexapeptides able to form polymeric ␤-sheet structures arranged in a similar fashion to that assu...

SummaryProtein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra-and pentaacylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing monophosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a b-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.