Chaperonin-assisted protein folding: a chronologue.

This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.

Hsp110 mitigates α-synuclein pathology in vivo.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.

Transfer of pathogenic and nonpathogenic cytosolic proteins between spinal cord motor neurons in vivo in chimeric mice.

Recent studies have reported spread of pathogenic proteins in the mammalian nervous system, but whether nonpathogenic ones spread is unknown. We initially investigated whether spread of a mutant amyotrophic lateral sclerosis-associated cytosolic superoxide dismutase 1 (SOD1) protein between motor neurons could be detected in intact chimeric mice. Eight-cell embryos from G85R SOD1YFP and G85R SOD1CFP mice were aggregated, and spinal cords of adult chimeric progeny were examined for motor neurons with cytosolic double fluorescence. By 3 mo of age, we observed extensive double fluorescence, including in amyotrophic lateral sclerosis-affected cranial nerve motor nuclei but not in the relatively spared extraocular nuclei. Chimeras of nonpathogenic wtSOD1YFP and G85R SOD1CFP also exhibited double fluorescence. In a third chimera, mitochondrial mCherry did not transfer to G85R SOD1YFP motor neurons, suggesting that neither RNA nor organelles transfer, but mito-mCherry neurons received G85R SOD1YFP. In a chimera of ChAT promoter-EGFP and mito-mCherry, EGFP efficiently transferred to mito-mCherry+ cells. Thus, nonpathogenic cytosolic proteins appear capable of transfer. During study of both the SOD1FP and EGFP chimeras, we observed fluorescence also in small cells neighboring the motor neurons, identified as mature gray matter oligodendrocytes. Double fluorescence in the G85R SOD1FP chimera and observation of the temporal development of fluorescence first in motor neurons and then in these oligodendrocytes suggest that they may be mediators of transfer of cytosolic proteins between motor neurons.

Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110.

Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoter-driven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ∼5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery.

Absence of lipofuscin in motor neurons of SOD1-linked ALS mice.

Lipofuscin, or aging pigment, is accreted as red autofluorescence in the lysosomes of motor neuron cell bodies in the ventral horn of WT mice by 3 mo of age. Strikingly, in two presymptomatic ALS mouse strains transgenic for mutant human Cu/Zn superoxide dismutase (SOD1), G85R SOD1YFP and G93A SOD1, little or no lipofuscin was detected in motor neuron cell bodies. Two markers of autophagy, sequestosome 1 (SQSTM1/p62) and microtubule-associated protein 1 light chain 3 (LC3), were examined in the motor neuron cell bodies of G85R SOD1YFP mice and found to be reduced relative to WT SOD1YFP transgenic mice. To elucidate whether the autophagy/lysosome pathway was either impaired or hyperactive in motor neurons, chloroquine was administered to 3-mo-old G85R SOD1YFP mice to block lysosomal hydrolysis. After 2 wk, lipofuscin was now observed in motor neurons, and SQSTM1 and LC3 levels approached those of WT SOD1YFP mice, suggesting that the autophagy/lysosome pathway is hyperactive in motor neurons of SOD1-linked ALS mice. This seems to be mediated at least in part through the mammalian target of rapamycin complex 1 (MTORC1) pathway, because levels of Ser757-phosphorylated Unc-51-like kinase 1 (ULK1), an MTORC1 target, were greatly reduced in the G85R SOD1YFP motor neurons, correspondent to an activated state of ULK1 that initiates autophagy.

Production of RNA for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

RNA-Seq profiling of spinal cord motor neurons from a presymptomatic SOD1 ALS mouse.

Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ~10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5-6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.

Structure and allostery of the chaperonin GroEL.

Chaperonins are intricate allosteric machines formed of two back-to-back, stacked rings of subunits presenting end cavities lined with hydrophobic binding sites for nonnative polypeptides. Once bound, substrates are subjected to forceful, concerted movements that result in their ejection from the binding surface and simultaneous encapsulation inside a hydrophilic chamber that favors their folding. Here, we review the allosteric machine movements that are choreographed by ATP binding, which triggers concerted tilting and twisting of subunit domains. These movements distort the ring of hydrophobic binding sites and split it apart, potentially unfolding the multiply bound substrate. Then, GroES binding is accompanied by a 100° twist of the binding domains that removes the hydrophobic sites from the cavity lining and forms the folding chamber. ATP hydrolysis is not needed for a single round of binding and encapsulation but is necessary to allow the next round of ATP binding in the opposite ring. It is this remote ATP binding that triggers dismantling of the folding chamber and release of the encapsulated substrate, whether folded or not. The basis for these ordered actions is an elegant system of nested cooperativity of the ATPase machinery. ATP binds to a ring with positive cooperativity, and movements of the interlinked subunit domains are concerted. In contrast, there is negative cooperativity between the rings, so that they act in alternation. It is remarkable that a process as specific as protein folding can be guided by the chaperonin machine in a way largely independent of substrate protein structure or sequence.

Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice.

Mutations of cytosolic Cu/Zn superoxide dismutase 1 (SOD1) in humans and overexpression of mutant human SOD1 genes in transgenic mice are associated with the motor neuron degenerative condition known as amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease). Gain-of-function toxicity from the mutant protein expressed in motor neurons, associated with its misfolding and aggregation, leads to dysfunction and cell death, associated with paralyzing disease. Here, using hydrogen-deuterium exchange in intact mice in vivo, we have addressed whether an ALS-associated mutant protein, G85R SOD1-YFP, is subject to the same rate of turnover in spinal cord both early in the course of the disease and later. We find that the mutant protein turns over about 10-fold faster than a similarly expressed wild-type fusion and that there is no significant change in the rate of turnover as animals age and disease progresses.

Double mutant MBP refolds at same rate in free solution as inside the GroEL/GroES chaperonin chamber when aggregation in free solution is prevented.

Under "permissive" conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5-10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.

ATP-triggered ADP release from the asymmetric chaperonin GroEL/GroES/ADP7 is not the rate-limiting step of the GroEL/GroES reaction cycle.

The GroEL/GroES protein folding chamber is formed and dissociated by ATP binding and hydrolysis. ATP hydrolysis in the GroES-bound (cis) ring gates entry of ATP into the opposite unoccupied trans ring, which allosterically ejects cis ligands. While earlier studies suggested that hydrolysis of cis ATP is the rate-limiting step of the cycle (t1/2 approximately 10 s), a recent study suggested that ADP release from the cis ring may be rate-limiting (t1/2 approximately 15-20 s). Here we have measured ADP release using a coupled enzyme assay and observed a t1/2 for release of

GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state.

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-of-addition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.

The GroEL/GroES cis cavity as a passive anti-aggregation device.

The GroEL/GroES chaperonin folding chamber is an encapsulated space of approximately 65 A diameter with a hydrophilic wall, inside of which many cellular proteins reach the native state. The question of whether the cavity wall actively directs folding reactions or is playing a passive role has been open. We review past and recent observations and conclude that the chamber functions as a passive "Anfinsen cage" that prevents folding monomers from multimolecular aggregation.

Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding.

The chaperonin ring assembly GroEL provides kinetic assistance to protein folding in the cell by binding non-native protein in the hydrophobic central cavity of an open ring and subsequently, upon binding ATP and the co-chaperonin GroES to the same ring, releasing polypeptide into a now hydrophilic encapsulated cavity where productive folding occurs in isolation. The fate of polypeptide during binding, encapsulation, and folding in the chamber has been the subject of recent experimental studies and is reviewed and considered here. We conclude that GroEL, in general, behaves passively with respect to its substrate proteins during these steps. While binding appears to be able to rescue non-native polypeptides from kinetic traps, such rescue is most likely exerted at the level of maximizing hydrophobic contact, effecting alteration of the topology of weakly structured states. Encapsulation does not appear to involve 'forced unfolding', and if anything, polypeptide topology is compacted during this step. Finally, chamber-mediated folding appears to resemble folding in solution, except that major kinetic complications of multimolecular association are prevented.

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state.

Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a "power stroke" of forceful apical movement to eject polypeptide into the chamber.

Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin cavity and free in solution.

The chaperonin GroEL binds non-native polypeptides in an open ring via hydrophobic contacts and then, after ATP and GroES binding to the same ring as polypeptide, mediates productive folding in the now hydrophilic, encapsulated cis chamber. The nature of the folding reaction in the cis cavity remains poorly understood. In particular, it is unclear whether polypeptides take the same route to the native state in this cavity as they do when folding spontaneously free in solution. Here, we have addressed this question by using NMR measurements of the time course of acquisition of amide proton exchange protection of human dihydrofolate reductase (DHFR) during folding in the presence of methotrexate and ATP either free in solution or inside the stable cavity formed between a single ring variant of GroEL, SR1, and GroES. Recovery of DHFR refolded by the SR1/GroES-mediated reaction is 2-fold higher than in the spontaneous reaction. Nevertheless, DHFR folding was found to proceed by the same trajectories inside the cis folding chamber and free in solution. These observations are consistent with the description of the chaperonin chamber as an "Anfinsen cage" where polypeptide folding is determined solely by the amino acid sequence, as it is in solution. However, if misfolding occurs in the confinement of the chaperonin cavity, the polypeptide chain cannot undergo aggregation but rather finds its way back to a productive pathway in a manner that cannot be accomplished in solution, resulting in the observed high overall recovery.

Two families of chaperonin: physiology and mechanism.

Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding?

Perturbed ATPase activity and not "close confinement" of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL.

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of "dwell time" for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the approximately 10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of "close confinement" on folding in the cis cavity.

Disulfide formation as a probe of folding in GroEL-GroES reveals correct formation of long-range bonds and editing of incorrect short-range ones.

The chaperonin GroEL assists protein folding by binding nonnative forms through exposed hydrophobic surfaces in an open ring and mediating productive folding in an encapsulated hydrophilic chamber formed when it binds GroES. Little is known about the topology of nonnative proteins during folding inside the GroEL-GroES cis chamber. Here, we have monitored topology employing disulfide bond formation of a secretory protein, trypsinogen (TG), that behaves in vitro as a stringent, GroEL-GroES-requiring substrate. Inside the long-lived cis chamber formed by SR1, a single-ring version of GroEL, complexed with GroES, we observed an ordered formation of disulfide bonds. First, short-range disulfides relative to the primary structure formed, both native and nonnative. Next, the two long-range native disulfides that "pin" the two beta-barrel domains together formed. Notably, no long-range nonnative bonds were ever observed, suggesting that a native-like long-range topology is favored. At both this time and later, however, the formation of several medium-range nonnative bonds mapping to one of the beta-barrels was observed, reflecting that the population of local nonnative structure can occur even within the cis cavity. Yet both these and the short-range nonnative bonds were ultimately "edited" to native, as evidenced by the nearly complete recovery of native TG. We conclude that folding in the GroEL-GroES cavity can favor the formation of a native-like topology, here involving the proper apposition of the two domains of TG; but it also involves an ATP-independent conformational "editing" of locally incorrect structures produced during the dwell time in the cis cavity.