Environment-transformable sequence-structure relationship: a general mechanism for proteotoxicity

Abstract

In his Nobel Lecture, Anfinsen stated "the native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment." As aqueous solutions and membrane systems co-exist in cells, proteins are classified into membrane and non-membrane proteins, but whether one can transform one into the other remains unknown. Intriguingly, many well-folded non-membrane proteins are converted into "insoluble" and toxic forms by aging- or disease-associated factors, but the underlying mechanisms remain elusive. In 2005, we discovered a previously unknown regime of proteins seemingly inconsistent with the classic "Salting-in" dogma: "insoluble" proteins including the integral membrane fragments could be solubilized in the ion-minimized water. We have thus successfully studied "insoluble" forms of ALS-causing P56S-MSP, L126Z-SOD1, nascent SOD1 and C71G-Profilin1, as well as E. coli S1 fragments. The results revealed that these "insoluble" forms are either unfolded or co-exist with their unfolded states. Most unexpectedly, these unfolded states acquire a novel capacity of interacting with membranes energetically driven by the formation of helices/loops over amphiphilic/hydrophobic regions which universally exit in proteins but are normally locked away in their folded native states. Our studies suggest that most, if not all, proteins contain segments which have the dual ability to fold into distinctive structures in aqueous and membrane environments. The abnormal membrane interaction might initiate disease and/or aging processes; and its further coupling with protein aggregation could result in radical proteotoxicity by forming inclusions composed of damaged membranous organelles and protein aggregates. Therefore, environment-transformable sequence-structure relationship may represent a general mechanism for proteotoxicity.

Keywords: Aging; Liquid–liquid phase separation (LLPS); Membrane interaction; Neurodegenerative diseases; Prion-like domains; Proteotoxicity.

Fig. 1

Sequence–structure relationship of proteins. Based on the sequences as represented by five types of amino acids, proteins can be classified into high-complexity or random (a), and low-complexity or non-random (f) sequences. A portion of proteins of the high-complexity sequence can fold into uniquely folded structures soluble in vivo with high concentrations of salts (c), while many of proteins of the low-complexity sequence remain intrinsically disordered (e). Interestingly, a large amount of proteins of both high- and low-complexity sequences appear to be aggregation-prone or even insoluble in vivo with high concentrations of salts (d). Furthermore, ~30% proteins of both high- and low-complexity sequences can fold in the membrane environments (b)

Fig. 2

Mechanism for the prion-like domains to self-assemble into liquid droplets and fibril structures. a The self-assembly of the prion-like domain dominated by aromatic residues as exemplified by the FUS prion-like domain. The monomeric prion-like domain enriched in polar and uncharged residues (Ser, Thr, Asn, Gln and aromatic residues such as Tyr) exists in equilibrium between two conformational states: I under some conditions such as at low pH, the majority of Ser, Thr, Asn and Gln side chains are involved in forming hydrogen bonds with the backbone atoms; and consequently the backbone adopts a conformation with many dynamic loops/turns; II at neutral pH some hydrogen bonds become disrupted due to the rapid dissociation of the backbone amide protons, and consequently the side chains are liberated and the backbone adopts a more extended conformation. On the one hand, both states regardless at low pH (I) or neutral pH (II) can self-assemble into liquid droplets (III and IV) in which aromatic residues at strategical position provide relatively strong interactions to form dynamic oligomers with the backbone largely disordered which are sufficient to trigger liquid-liquid phase separation. On the other hand, only the state IV with a large portion of the Ser, Thr, Asn and Gln side-chains liberated will be further exaggerated into forming fibril structures with cross-β structures stabilized by “hydrogen-bond/polar/steric zippers” (V) under certain conditions such as long incubation. b The self-assembly of the prion-like domain dominated by hydrophobic residues as exemplified by the TDP-43 prion-like domain. The monomeric prion-like domain enriched in polar and uncharged residues (Ser, Thr, Asn and Gln) and further containing a hydrophobic region also exists in equilibrium between two conformational states I and II. Both states can also self-assemble into liquid droplets (III and IV) in which the formation of dynamic oligomers is mainly mediated by the self-association over the hydrophobic region. The state IV will be further exaggerated into forming fibril structures over the Ser, Thr, Asn and Gln rich region stabilized by “hydrogen-bond/polar/steric zippers” (V). Remarkably, the hydrophobic region remains to be helical even in the fibrils of the wild-type TDP-43 prion-like domain, while it transforms into the classic amyloid structure stabilized by hydrophobic interactions in the fibrils of the ALS-causing mutants. c The atomic structure of the fibrils formed by the FUS prion-like domain over residues 39–95 (I), which is characterized by the intrinsic visible fluorescence with the emission maximum at ~455 nm (II). d The atomic structure of the classic amyloid fibrils formed by Aβ42 (I), which is characterized by the intrinsic visible fluorescence with the emission maximum at ~475 nm (II). Green polar/charged residues, gray hydrophobic residues and pink aromatic residues

Fig. 3

Solution conformations of insoluble proteins solubilized in unsalted water. Four groups of conformations have been observed so far on the insoluble proteins solubilized in unsalted water: I those without stable secondary and tertiary structures; II those with secondary but without tertiary structures; III those with secondary structures as well as dynamic tertiary packing which is molten globule-like; and IV those with coexistence between the folded and unfolded states in equilibrium

Fig. 4

Transformation of a well-folded and soluble cytosolic protein into membrane-interacting form. a By disease- and aging-associated factors, a well-folded and highly soluble cytosolic protein (I) can be converted into a highly disordered form, which is only soluble in salt-minimized water (II). As this highly disordered form universally contains hydrophobic, and/or amphiphilic patches, it acquires the novel capacity to interact with membranes energetically driven by folding into non-native helix/loop structures in membranes (III). b Residues 28–38 (in blue) of SOD1 adopt a β-strand in the native structure (I), but become disordered in the ALS-causing L126Z-SOD1 (II). However, these residues transform into a helix in the membrane-embedded L126Z-SOD1 (III). Furthermore, eight isolated fragments of SOD1 (28–28) can assemble into an amyloid structure after incubation (IV)

Fig. 5

Mechanisms for proteins characterized by aggregation to manifest proteotoxicity by abnormally interacting with membranes. The disordered protein with significant exposure of hydrophobic and/or amphiphilic patches is only soluble in salt-minimized water (a), but become severely aggregated or even insoluble in vivo with ~150 mM salts (b), which might be degraded by complex machineries such as autophagosome-lysosome pathway (c). However, before it becomes completely insoluble, or under some conditions even the formed inclusions and amyloid fibrils might be disassembled into soluble oligomers, it can abnormally interact with membranes (d) by three mechanisms: I direct disruption of membrane structures and dynamics such as by forming ion channels/pores or large aggregates within membranes; and/or II perturbation/initiation of membrane-anchored signal pathways or machineries such as ion channels; and/or III interference in the membrane remodeling such as budding, shaping, trafficking and fusion. In particular, the coupled abilities of such a protein to aggregate and interact with membranes might radically damage various membranous organelles to manifest its proteotoxicity by forming inclusions composed of damaged membranous organelles and protein (e)