Jun-Xia Lu

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) regulates cell death pathways through RHIM domain–mediated amyloid fibril formation. While amyloid fibrils typically exist as single filaments, we identified a higher-order architecture—the mouse RIPK1 (mRIPK1) fibril network—formed by the self-assembly of mRIPK1 fibrils into quadrilateral/hexagonal lattices under slightly acidic conditions. Using an integrative approach combining solid-state NMR, transmission electron microscopy, atomic force microscopy, X-ray diffraction, Cryo–electron tomography, and molecular dynamics simulations, we elucidated the atomic structure and assembly mechanism of this network. In this study, solid-state NMR analysis demonstrates that the mRIPK1 fibril core adopts an N-shaped parallel β-sheet conformation, with dynamic regions flanking the fibril core that likely participate in network formation. We propose that the electrostatic interactions between fibril core edge residues D516-K519 or D537-K519 are essential for the network formation, with proper positioning and exposure for interaction determined by the fibril structure and the length of the dynamic flexible domain, particularly the periodic twist of the fibril. Site-directed mutagenesis confirms the critical role of these edge residues in maintaining network integrity. This study presents an atomic model of a higher-order assembly formed by naturally occurring amyloid fibrils, offers fundamental insights into hierarchical fibril assembly, and establishes a framework for designing engineered amyloid-based materials.

Osteoporosis (OP), which is a common skeletal disease with different causes, is prevalent in the aging population. Postmenopause women generally suffer from OP with bone loss due to estrogen deficiency. Diabetes is also associated with OP by complex metabolic mechanisms. Bone qualities of OP caused by aging were compared with those of the ovariectomy (OVX) model and the Type 2 diabetic model using Sprague-Dawley (SD) rats in our study. Combining with micro-computed tomography (μ-CT) and solid-state NMR (SSNMR) methods, this research studied bone changes in SD rats from tissue level to the molecular level. The studies revealed bone loss was most significant for cancellous bones but not for cortical bones in OP rats. However, at the molecular level, the content of HAP in cortical bone increased with aging, contributing to the brittleness of the bone. Triglyceride, as a senescence maker of osteocyte in cortical bone, was also identified to be closely associated with OP in aging and OVX rats but not in diabetic rats. This research suggests that changes of bone quality at the molecular level more objectively reflect the bone tissue reconstruction of OP with various causes rather than mere bone loss revealed by μ-CT analysis.

Osteoporosis (OP) which is a common skeletal disease with different causation is prevalent in aging population. Postmenopause women generally suffer from OP with bone loss due to estrogen deficiency. Diabetes are also associated with OP by complex metabolic mechanisms. Bone qualities of OP caused by aging were compared with the ovariectomy (OVX) model and the Type 2 diabetic model using Sprague-Dawley (SD) rats in our study. Combining with micro-computed tomography ( μ -CT) and solid-state NMR (SSNMR) methods, this research studied bone changes in SD rats from tissue level to the molecular level. The studies revealed bone loss was most significant for cancellous bones, but not for cortical bones in OP rats. However, at the molecular level, the content of HAP in cortical bone increased with aging, contributing to the brittleness of the bone. Triglyceride, as a senescence maker of osteocyte in cortical bone, was also identified to be closely associated with OP in aging and OVX rats, but not in diabetic rats. This research suggests differing bone qualities in molecular level of OP with various causes more objectively reflect the bone tissue reconstruction rather than bone loss in μ -CT analysis.

Receptor-interacting protein kinase 1 (RIPK1) is a therapeutic target in treating neurodegenerative diseases and cancers. RIPK1 has three distinct functional domains, with the center domain containing a receptor-interacting protein homotypic interaction motif (RHIM), which mediates amyloid formation. The functional amyloid formed by RIPK1 and/or RIPK3 is a crucial intermediate in regulating cell necroptosis. In this study, the amyloid structure of mouse RIPK1, formed by an 82-residue sequence centered at RHIM, is presented. It reveals the “N”-shaped folding of the protein subunit in the fibril with four β-strands. The folding pattern is shared by several amyloid structures formed by proteins with RHIM, with the central β-strand formed by the most conserved tetrad sequence I/VQI/VG. However, the solid-state NMR results indicate a structural difference between mouse RIPK1 and mouse RIPK3. A change in the structural rigidity is also suggested by the observation of weakened signals for mouse RIPK3 upon mixing with RIPK1 to form the RIPK1/RIPK3 complex fibrils. Our results provide vital information to understand the interactions between different proteins with RHIM, which will help us further comprehend the regulation mechanism in cell necroptosis.

Some proteins perform their biological functions by changing their material states through liquid-liquid phase separation. Upon phase separation, the protein condenses into a concentrated liquid phase and sometimes into a gel phase, changing its dynamic properties and intermolecular interactions, thereby regulating cellular functions. Although the biological significance of this phenomenon has been widely recognized by researchers, there is still a lack of a comprehensive understanding of the structural and dynamic properties of the protein in the condensed phase. In this phase, molecules usually contain domains with varied dynamic properties and undergo intermediate exchanges. Magic angle spinning (MAS) solid-state NMR (SSNMR) experiments are very powerful in studying rigid protein polymers such as amyloid. The incorporation of solution-like experiments into SSNMR and the development of J-coupling based MAS SSNMR techniques extend its ability to study partially mobile segments of proteins in a condensed liquid or gel phase which are not visible by solution NMR or dipolar-coupling based SSNMR. Therefore, it has been applied in studying protein condensation and has provided very important information that is hard to obtain by other techniques.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed ‘functional amyloids’, contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme — which we name as ‘amyloidase’ — that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X₁)φ(X₃). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8’s amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

Corrigendum: Calcium interactions in amelogenin-derived peptide assembly

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between ³¹P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR ³¹P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register β-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.