Metal Binding Properties of the N-Terminus of the Functional Amyloid Orb2.

Thalia H Bajakian,Maria A Soria,Ansgar B Siemer,Ji Yun Kim,Rachel J Service,Maïwenn Beaugrand,Silvia A Cervantes

doi:10.3390/biom7030057

Thalia H Bajakian, Maria A Soria + Show 5 more

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https://doi.org/10.3390/biom7030057

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Journal: Biomolecules	Publication Date: Aug 1, 2017
Citations: 4	License type: CC BY 4.0

Affiliation: McGill University

Abstract

The cytoplasmic polyadenylation element binding protein (CPEB) homologue Orb2 is a functional amyloid that plays a key regulatory role for long-term memory in Drosophila. Orb2 has a glutamine, histidine-rich (Q/H-rich) domain that resembles the Q/H-rich, metal binding domain of the Hpn-like protein (Hpnl) found in Helicobacter pylori. In the present study, we used chromatography and isothermal titration calorimetry (ITC) to show that the Q/H-rich domain of Orb2 binds Ni2+ and other transition metals ions with μM affinity. Using site directed mutagenesis, we show that several histidine residues are important for binding. In particular, the H61Y mutation, which was previously shown to affect the aggregation of Orb2 in cell culture, completely inhibited metal binding of Orb2. Finally, we used thioflavin T fluorescence and electron microscopy images to show that Ni2+ binding induces the aggregating of Orb2 into structures that are distinct from the amyloid fibrils formed in the absence of Ni2+. These data suggest that transition metal binding might be important for the function of Orb2 and potentially long-term memory in Drosophila.

Highlights

The aggregation of proteins into amyloid fibrils is thought to be a sporadic event in the case of amyloid diseases
In Aplysia, the neuronal isoform of cytoplasmic polyadenylation element binding protein (CPEB) (ApCPEB), which is a key regulator of long-term potentiation (LTP), is activated by undergoing a conformational change from a soluble, oligomeric to an aggregated, amyloid-like state [2,3]
We showed that the same N-terminal region of Orb2A can bind to negatively charged lipid vesicles and subsequently form a helical structure that prevents the formation of amyloid fibrils [15]

Summary

Introduction

The aggregation of proteins into amyloid fibrils is thought to be a sporadic event in the case of amyloid diseases. To understand how the formation of functional amyloid is regulated, we are studying factors that induce and prevent the aggregation of the functional amyloid Orb from. Orb belongs to the class of cytoplasmic polyadenylation element binding proteins (CPEBs), which are extranuclear regulators of protein expression [1]. In Aplysia, the neuronal isoform of CPEB (ApCPEB), which is a key regulator of long-term potentiation (LTP), is activated by undergoing a conformational change from a soluble, oligomeric to an aggregated, amyloid-like state [2,3]. Orb is a CPEB homologue in Drosophila melanogaster which acts as a key regulator of synapse specific protein expression that is important for maintaining memories beyond 24 h [6,7,8,9,10,11].

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