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.

A specific group of transmembrane receptors, including the β1-adrenergic receptor (β1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the β1-AR, and the C-terminal domain of lamellipodin (LPD). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets, and LPD also phase separates with the actin polymerase VASP. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD C-terminal domain. Our results demonstrate how the multivalent interactions between endophilin, LPD, and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important issue. One mechanism entails the generation of force by the nascent protein. Cotranslational processes, such as nascent protein folding, the emergence of unfolded nascent chain segments from the ribosome’s exit tunnel, and insertion of the nascent chain into or translocation of the nascent chain through membranes, can generate forces that are transmitted back to the peptidyl transferase center and affect translation rates. In this Perspective, we examine the processes that generate these forces, the mechanisms of transmission along the ribosomal exit tunnel to the peptidyl transferase center, and the effects of force on the ribosome’s catalytic cycle. We also discuss the physical models that have been developed to predict and explain force generation for individual processes and speculate about other processes that may generate forces that have yet to be tested.

Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin’s BAR domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per µm2 represented by a covalently conjugated TIL region from β1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.

Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, 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 find that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be degraded. These misfolded states can persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, allowing them to bypass the proteostasis machinery and processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. These results explain how proteins misfold into soluble, non-functional conformations that bypass cellular quality controls, and indicate that, unexpectedly, this is a wide-spread cellular phenomenon that can lead to reduced protein function across the cytosolic proteome. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

Endocytosis of transmembrane receptors initiates via molecular interactions between the activated receptor and the endocytic machinery. A specific group of receptors, including the β1-adrenergic receptor (β1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the β1-AR, and the proline-rich-motifs of lamellipodin (LPD-PRMs). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD-PRMs. Our results demonstrate how the multivalent interactions between endophilin, LPD-PRMs and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

The Bin/Amphiphysin/Rvs (BAR) family protein endophilin plays key roles in membrane curvature generation during endocytosis of cellular receptors. The Src homology 3 (SH3) domain of endophilin interacts with the proline rich third intracellular loop (TIL) of various G-protein coupled receptors (GPCRs). While electrostatic interactions between BAR domain and anionic membrane lipids have been considered to be the major driving force in curvature generation, it is unclear how the direct interaction between TIL and SH3 affects this function and its coupling with receptor internalization. Here we show that TIL mediated interactions alone not only recruit endophilin to the membrane but also facilitate curvature sorting and curvature generating behavior of endophilin. To demonstrate this, we designed model membranes with covalently lipid-conjugated TIL and lipids without net negative charge so that endophilin was recruited exclusively via SH3/TIL interactions. We find curvature generation and curvature sorting under those conditions. Furthermore, we show that TIL interacts electrostatically with membranes in the presence of anionic lipids and that this interaction can interfere with binding of SH3. Overall, our study suggests that an interplay between TIL, charged membranes, BAR domain, and SH3 domain mediate membrane curvature generation to regulate receptor endocytosis following receptor stimulation.

No abstract