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Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing β-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only inregister β-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register β-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register β-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway. [ABSTRACT FROM AUTHOR]

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function. [ABSTRACT FROM AUTHOR]

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid-liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect-based chemicalenrichment method (SECEM) coupled with liquid chromatography-tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG-rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG-rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics. [ABSTRACT FROM AUTHOR]

Heterozygous point mutations of α-synuclein (a-syn) have been linked to the early onset and rapid progression of familial Parkinson's diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) a-syn and its role in the exacerbated pathology of a-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant a-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different a-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryoelectron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT a-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT a-syn in such a way that the mutant pathology would be amplified in fPD. [ABSTRACT FROM AUTHOR]

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three ß-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation. [ABSTRACT FROM AUTHOR]

Membraneless organelles contain a wide spectrum of molecular chaperones, indicating their important roles in modulating the metastable conformation and biological function of membraneless organelles. Here we report that class I and II Hsp40 (DNAJ) proteins possess a high ability of phase separation rendered by the flexible G/F-rich region. Different Hsp40 proteins localize in different membraneless organelles. Specifically, human Hdj1 (DNAJB1), a class II Hsp40 protein, condenses in ubiquitin (Ub)-rich nuclear bodies, while Hdj2 (DNAJA1), a class I Hsp40 protein, condenses in nucleoli. Upon stress, both Hsp40 proteins incorporate into stress granules (SGs). Mutations of the G/F-rich region not only markedly impaired Hdj1 phase separation and SG involvement and disrupted the synergistic phase separation and colocalization of Hdj1 and fused in sarcoma (FUS) in cells. Being cophase separated with FUS, Hdj1 stabilized the liquid phase of FUS against proceeding into amyloid aggregation in vitro and alleviated abnormal FUS aggregation in cells. Moreover, Hdj1 uses different domains to chaperone FUS phase separation and amyloid aggregation. This paper suggests that phase separation is an intrinsic property of Hsp40 proteins, which enables efficient incorporation and function of Hsp40 in membraneless organelles and may further mediate the buildup of chaperone network in membraneless organelles. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson’s disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Using and engineering amyloid as nanomaterials are blossoming trends in bionanotechnology. Here, we show our discovery of an amyloid structure, termed "amyloid-like nanosheet," formed by a key amyloid-forming segment of Alzheimer's Aβ. Combining multiple biophysical and computational approaches, we proposed a structural model for the nanosheet that is formed by stacking the amyloid fibril spines perpendicular to the fibril axis. We further used the nanosheet for laboratorial retroviral transduction enhancement and directly visualized the presence of virus on the nanosheet surface by electron microscopy. Furthermore, based on our structural model, we designed nanosheet-forming peptides with different functionalities, elucidating the potential of rational design for amyloid-based materials with novel architecture and function. [ABSTRACT FROM AUTHOR]

New materials capable of binding carbon dioxide are essential for addressing climate change. Here, we demonstrate that amyloids, self-assembling protein fibers, are effective for selective carbon dioxide capture. Solid-state NMR proves that amyloid fibers containing alkylamine groups reversibly bind carbon dioxide via carbamate formation. Thermodynamic and kinetic capture-and-release tests show the carbamate formation rate is fast enough to capture carbon dioxide by dynamic separation, undiminished by the presence of water, in both a natural amyloid and designed amyloids having increased carbon dioxide capacity. Heating to 100 °C regenerates the material. These results demonstrate the potential of amyloid fibers for environmental carbon dioxide capture. [ABSTRACT FROM AUTHOR]