HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

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.

Solid-state NMR studies of amyloids.

Amyloids have special structural properties and are involved in many aspects of biological function. In particular, amyloids are the cause or hallmarks of a group of notorious and incurable neurodegenerative diseases. The extraordinary high molecular weight and aggregation states of amyloids have posed a challenge for researchers studying them. Solid-state NMR (SSNMR) has been extensively applied to study the structures and dynamics of amyloids for the past 20 or more years and brought us tremendous progress in understanding their structure and related diseases. These studies, at the same time, helped to push SSNMR technical developments in sensitivity and resolution. In this review, some interesting research studies and important technical developments are highlighted to give the reader an overview of the current state of this field.

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 amyloid structure of mouse RIPK3 (receptor interacting protein kinase 3) in cell necroptosis.

RIPK3 amyloid complex plays crucial roles during TNF-induced necroptosis and in response to immune defense in both human and mouse. Here, we have structurally characterized mouse RIPK3 homogeneous self-assembly using solid-state NMR, revealing a well-ordered N-shaped amyloid core structure featured with 3 parallel in-register ß-sheets. This structure differs from previously published human RIPK1/RIPK3 hetero-amyloid complex structure, which adopted a serpentine fold. Functional studies indicate both RIPK1-RIPK3 binding and RIPK3 amyloid formation are essential but not sufficient for TNF-induced necroptosis. The structural integrity of RIPK3 fibril with three ß-strands is necessary for signaling. Molecular dynamics simulations with a mouse RIPK1/RIPK3 model indicate that the hetero-amyloid is less stable when adopting the RIPK3 fibril conformation, suggesting a structural transformation of RIPK3 from RIPK1-RIPK3 binding to RIPK3 amyloid formation. This structural transformation would provide the missing link connecting RIPK1-RIPK3 binding to RIPK3 homo-oligomer formation in the signal transduction.

RIP3-mediated necroptosis is regulated by inter-filament assembly of RIP homotypic interaction motif.

Necroptosis is mediated by signaling complexes called necrosomes, which contain receptor-interacting protein 3 (RIP3) and upstream effectors, such as RIP1. In necrosomes, the RIP homotypic interaction motif (RHIM) of RIP3 and RIP1 forms amyloidal complex. But how the amyloidal necrosomes control RIP3 activation and cell necroptosis has not been determined. Here, we showed that RIP3 amyloid fibrils could further assemble into large fibrillar networks which presents as cellular puncta during necroptosis. A viral RHIM-containing necroptosis inhibitor M45 could form heteroamyloid with RIP3 in cells and prevent RIP3 puncta formation and cell necroptosis. We characterized mutual antagonism between RIP3-RHIM and M45-RHIM in necroptosis regulation, which was caused by distinct inter-filament interactions in RIP3, M45 amyloids revealed with atomic force microscopy. Moreover, double mutations Asn464 and Met468 in RIP3-RHIM to Asp disrupted RIP3 kinase-dependent necroptosis. While the mutant RIP3(N464D/M468D) could form amyloid as wild type upon necroptosis induction. Based on these results, we propose that RIP3 amyloid formation is required but not sufficient in necroptosis signaling, the ordered inter-filament assembly of RIP3 is critical in RIP3 amyloid mediated kinase activation and cell necroptosis.

Principles Governing Catalytic Activity of Self-Assembled Short Peptides.

Molecular self-assembly provides a chemical strategy for the synthesis of nanostructures by using the principles of nature, and peptides serve as the promising building blocks to construct adaptable molecular architectures. Recently, a series of heptapeptides with alternative hydrophobic and hydrophilic residues were reported to form amyloid-like structures, which are capable of catalyzing acyl ester hydrolysis with remarkable efficiency. However, information remains elusive about the atomic structures of the fibrils. What is the origin of the sequence-dependent catalytic activity? How is the ester hydrolysis catalyzed by the fibrils? In this work, the atomic structures of the aggregates were determined by using molecular modeling and further validated by solid-state NMR experiments, where the fibril with high activity adopts twisted parallel configuration within each layer, and the one with low activity is in flat antiparallel configuration. The polymorphism originates from the interactions between different regions of the building block peptides, where the delicate balance between rigidity and flexibility plays an important role. We further show that the p-nitrophenylacetate ( pNPA) hydrolysis reactions catalyzed by two different fibrils follow a similar mechanism, and the difference in microenvironment at the active site between the natural enzyme and the present self-assembled fibrils should account for the discrepancy in catalytic activities. The present work provides understanding of the structure and function of self-assembled fibrils formed with short peptides at an atomic level and thus sheds new insight on designing aggregates with better functions.