Creating an Amyloid 'Kaleidoscope' Using Short Iodinated Peptides.

Amyloid fibrils formed by peptides with different sequences exhibit diversified morphologies, material properties and activities, making them valuable for developing functional bionanomaterials. However, the molecular understanding underlying the structural diversity of peptide fibrillar assembly at atomic level is still lacking. In this study, by using cryogenic electron microscopy, we first revealed the structural basis underlying the highly reversible assembly of 1 GFGGNDNFG9 (referred to as hnRAC1) peptide fibril. Furthermore, by installing iodine at different sites of hnRAC1, we generated a collection of peptide fibrils with distinct thermostability. By determining the atomic structures of the iodinated fibrils, we discovered that iodination at different sites of the peptide facilitates the formation of diverse halogen bonds and triggers the assembly of entirely different structures of iodinated fibrils. Finally, based on this structural knowledge, we designed an iodinated peptide that assembles into new atomic structures of fibrils, exhibiting superior thermostability, that aligned with our design. Our work provides an in-depth understanding of the atomic-level processes underlying the formation of diverse peptide fibril structures, and paves the way for creating an amyloid "kaleidoscope" by employing various modifications and peptide sequences to fine-tune the atomic structure and properties of fibrillar nanostructures.

Structural Insights of Fe3+ Induced α-synuclein Fibrillation in Parkinson's Disease.

Amyloid aggregation of α-synuclein (α-syn) in Lewy bodies (LBs) is the pathological hallmark of Parkinson's disease (PD). Iron, especially Fe3+, is accumulated in substantia nigra of PD patients and co-deposited with α-syn in LBs. However, how Fe3+ modulates α-syn fibrillation at molecular level remains unclear. In this study, we found that Fe3+ can promote α-syn fibrillation at low concentration while inhibit its fibrillation at high concentration. NMR titration study shows poor interaction between α-syn monomer and Fe3+. Instead, we found that Fe3+ binds to α-syn fibrils. By using cryo-electron microscopy (cryo-EM), we further determined the atomic structure of α-syn fibril in complex with Fe3+ at the resolution of 2.7 Å. Strikingly, two extra electron densities adjacent to His50 and Glu57 were observed as putative binding sites of Fe3+ and water molecules, suggesting that Fe3+ binds to the negative cleft of the fibril and stabilizes the fibril structure for promoting α-syn aggregation. Further mutagenesis study shows mutation of His50 abolishes the Fe3+-facilitated fibrillation of α-syn. Our work illuminates the structural basis of the interaction of Fe3+ and α-syn in both monomeric and fibrillar forms, and sheds light on understanding the pathological role of Fe3+ in α-syn aggregation in PD.

Cryo-EM structure of an amyloid fibril formed by full-length human SOD1 reveals its conformational conversion.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial and sporadic ALS. SOD1 fibrils formed in vitro share toxic properties with ALS inclusions. Here we produced cytotoxic amyloid fibrils from full-length apo human SOD1 under reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofilament with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3-55) and C-terminal segment (residues 86-153) with an intrinsic disordered segment. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight ß-strands (to form a ß-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen ß-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

Prefrontal brain function in patients with chronic insomnia disorder: A pilot functional near-infrared spectroscopy study.

Purpose: Insomnia is one of the most common diseases in elderly patients, which seriously affect the quality of life and psychological state of patients. The purpose of this study was to investigate the changes in the functional network pattern of the prefrontal cortex in patients with chronic insomnia disorder (CID) after taking drugs, using non-invasive and low-cost functional neuroimaging with multi-channel near-infrared spectroscopy (fNIRS). Methods: All subjects were assessed using the Pittsburgh Sleep Quality Index (PSQI), Hamilton Depression Scale (HAMD), Hamilton Anxiety Scale (HAMA), and fNIRS. The fNIRS assessment consists of two parts: the verbal fluency test (VFT) task state and the resting state, which assessed the differences in prefrontal activation and functional connectivity, respectively. Results: A total of 30 patients with chronic insomnia disorder (CID) and 15 healthy peers completed the study. During the VFT task, a significantly lower PFC activation was observed in patients with insomnia compared to the control group (P < 0.05). However, the PFC activation in patients taking medication was higher than in patients who did not receive medication. Functional connectivity analysis showed a weaker mean PFC channel connectivity strength in patients with CID who did not receive drug treatment. Drug treatment resulted in enhanced functional connectivity of the prefrontal lobe, especially the DLPFC and frontal poles. Conclusion: A weak prefrontal cortex response was detected in patients with CID when performing the VFT task, which could be enhanced by taking hypnotics. The weakened right prefrontal lobe network may play a role in the development of CID. fNIRS may serve as a potential tool to assess sleep status and guide drug therapy.

O-Glycosylation Induces Amyloid-ß To Form New Fibril Polymorphs Vulnerable for Degradation.

Brain accumulation of amyloid-ß (Aß) peptides (resulting from a disrupted balance between biosynthesis and clearance) occurs during the progression of Alzheimer's disease (AD). Aß peptides have diverse posttranslational modifications (PTMs) that variously modulate Aß aggregation into fibrils, but understanding the mechanistic roles of PTMs in these processes remains a challenge. Here, we chemically synthesized three homogeneously modified isoforms of Aß (1-42) peptides bearing Tyr10 O-glycosylation, an unusual PTM initially identified from the cerebrospinal fluid samples of AD patients. We discovered that O-glycans significantly affect both the aggregation and degradation of Aß42. By combining cryo-EM and various biochemical assays, we demonstrate that a Galß1-3GalNAc modification redirects Aß42 to form a new fibril polymorphic structure that is less stable and more vulnerable to Aß-degrading enzymes (e.g., insulin-degrading enzyme). Thus, beyond showing how particular O-glycosylation modifications affect Aß42 aggregation at the molecular level, our study provides powerful experimental tools to support further investigations about how PTMs affect Aß42 fibril aggregation and AD-related neurotoxicity.

Genetic prion disease-related mutation E196K displays a novel amyloid fibril structure revealed by cryo-EM.

Prion diseases are caused by the conformational conversion of prion protein (PrP). Forty-two different mutations were identified in human PrP, leading to genetic prion diseases with distinct clinical syndromes. Here, we report the cryo­electron microscopy structure of an amyloid fibril formed by full-length human PrP with E196K mutation, a genetic Creutzfeldt-Jakob disease­related mutation. This mutation disrupts key interactions in the wild-type PrP fibril, forming an amyloid fibril with a conformation distinct from the wild-type PrP fibril and hamster brain­derived prion fibril. The E196K fibril consists of two protofibrils. Each subunit forms five ß strands stabilized by a disulfide bond and an unusual hydrophilic cavity stabilized by a salt bridge. Four pairs of amino acids from opposing subunits form four salt bridges to stabilize the zigzag interface of the two protofibrils. Our results provide structural evidences of the diverse prion strains and highlight the importance of familial mutations in inducing different strains.

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3.

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.

The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure.

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-ß2 (Kapß2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapß2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapß2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.