Molecular Determinants of Liquid Demixing and Amyloidogenesis in Human CPEB3

Abstract

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), is an RNA-binding protein which in its soluble state is localized in membraneless neuronal RNA granules keeping target mRNAs in a repressed state. The stimulus-dependent aggregation of CPEB3 activates target mRNAs translation, a central event for the maintenance of long-term memory-related synaptic plasticity in mammals. To date, the molecular determinants that govern both connected events remain unclear. Here, to gain insight into these processes, the biophysical properties of the human CPEB3 (hCPEB3) are characterized. We found that hCPEB3 homotypic condensation is mainly driven by hydrophobic interactions and occurs under physiological conditions. Moreover, hCPEB3 biomolecular condensates are dynamic inside living cells, whose localization and stabilization are mediated by its RNA-recognition domains.In contrast, the hCPEB3 polar N-terminal region is crucial for hCPEB3 amyloid-like aggregation in vitro, which is disrupted by the polyglutamine binding peptide 1 (QBP1), Aβ42 seeds and Hsp70, highlighting the importance of the Q4RQ4 tract as well as the hydrophobic residues for hCPEB3 functional aggregation. Based on these findings, we postulate a model for hCPEB3's role in memory persistence that advances a rather sophisticated control for hCPEB3 condensate dissociation and amyloid-like formation to achieve its physiological function.hCPEB3's demixing and amyloidogenesis Highlights • hCPEB3 forms toxic intermediates that persist longer than in other functional amyloids.• RNA-recognition domains stabilize hCPEB3 granule formation and dynamics.• Different segments within hCPEB3 promote amyloidogenesis and liquid demixing.• hCPEB3 amyloid formation requires both hydrophobic and polyQ segments. Graphical AbstracthCPEB3's demixing and amyloidogenesis

The role of CPEB family proteins in the nervous system function in the norm and pathology

“…The mouse and human CPEB3 proteins are homologs with 89% sequence identity. In human CPEB3, polyalanine and polyglutamine tracts are required for the induction of aggregation [ 24 ]. A high frequency of proline in the mammalian CPEB3 prion domain restricts amyloidogenesis in aggregation-prone regions of the protein to prevent excessive oligomerization, which can damage neurons.…”

“…Phosphorylated serine residues might be necessary for controlling the transition from the monomeric to an oligomeric form. In contrast to Orb2, functional amyloids formed by human CPEB3 do not differ from pathological amyloids, as shown by in vitro experiments [ 24 ]. Both functional and pathological amyloids form toxic metastable oligomer species.…”

“…However, toxic intermediates of functional amyloids are extremely transient, while toxic conformers of pathological amyloids are long-lived, lasting weeks. Metastable CPEB3 toxic oligomers were long-lived, similar to pathological amyloids [ 24 ]. The cytotoxic effect of CPEB3 oligomers was confirmed in a neuroblastoma cell culture.…”

Preferred conformations in the disordered region of human CPEB3, a functional amyloid linked to memory consolidation

“…The 426-residue long IDR of hCPEB3 plays a key role mediating memory persistence through this prion-like mechanism. It contains an amyloid-forming region spanning residues 1-200, and a condensate-promoting region formed by residues 250-426 which are linked by an alanine rich segment [22]. The full IDR is followed by two folded RRM which bind RNA and finally a ZZ-type ZnF domain ( Figure 1B ).…”

Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation

“…Whereas Aplysia and mammalian CPEB homologs share a similar domain organization with Drosophila Orb2, they lack the multiple His residues interspersed in their Q-rich segments (40,43,44) which are found in Orb2. This suggests that the pHregulated nature of Orb2 amyloid formation and dissociation may be substituted by other mechanisms.…”