Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional

Abstract

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

G-quadruplexes rescuing protein folding

“…A common assumption made in biological experiments is that soluble protein is typically well-folded and active. However, several studies have pointed out that this is an oversimplification (Danielsson et al, 2015; Ebbinghaus et al, 2010; Nissley et al, 2022; Waztor et al, 2020), as appears to be the case for TagRFP675 when not enough chaperone capacity is available to aid its folding.…”

Mechanical regulation of talin through binding and history-dependent unfolding

“…This possible mechanism would involve kinetic trapping in a stable structure ( 34 ). Using molecular dynamics simulations, it was proposed that, if unfolded, then half of the cytosolic Escherichia coli proteins could exhibit subpopulations of misfolded conformations, which would be kinetically trapped, but without propensity to aggregate ( 35 ). These conformations are rarely encountered in vivo for freshly expressed proteins, where the cotranslational folding restricts the folding kinetics ( 36 ).…”

A Newly Identified Class of Protein Misfolding in All-atom Folding Simulations Consistent with Limited Proteolysis Mass Spectrometry

“…Only a small number of mechanisms intrinsic to a protein's primary structure are known to cause monomeric protein misfolding (Table 1). Recently, high-throughput, coarse-grained simulations of protein synthesis and folding of the E. coli proteome have suggested there exists an additional widespread mechanism of misfolding [1][2][3] . Proteins can populate off-pathway misfolded states that involve a change in entanglement 1 .…”

“…The misfolded states observed in the aforementioned simulations involved either the gain of a non-native entanglement (i.e., the formation of an entanglement that is not present in the native ensemble, Table S1) or the loss of a native entanglement (i.e., an entanglement present in the native state fails to form, Table S1) [1][2][3] . This newly predicted class of misfolding offers an explanation for the decades old observations that non-functional protein molecules can persist for long-time scales in the presence of chaperones and not rapidly aggregate nor be degraded [7][8][9] .…”

“…Several mechanisms intrinsic to a protein's primary structure are known to cause monomeric protein misfolding (Table 1). Recently, high-throughput, coarse-grained simulations of protein synthesis and folding of the E. coli proteome have suggested there exists an additional widespread mechanism of misfolding [1][2][3] : proteins can populate off-pathway misfolded states that involve a change in non-covalent lasso entanglements 1,2 . This type of entanglement is defined by the presence of two structural components: a loop formed by a protein backbone segment closed by a non-covalent native contact, and another protein segment that is threaded through and around this loop [4][5][6] in some cases multiple times (Figs.…”