Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases.     

BRICHOS domain associated with lung fibrosis, dementia and cancer--a chaperone that prevents amyloid fibril formation?

The BRICHOS domain was initially defined from sequence alignments of the Bri protein associated with familial dementia, chondromodulin associated with chondrosarcoma and surfactant protein C precursor (proSP-C) associated with respiratory distress syndrome and interstitial lung disease (ILD). Today BRICHOS has been found in 12 protein families. Mutations in the Bri2 and proSP-C genes result in familial dementia and ILD, respectively, and both these conditions are associated with amyloid formation. Amyloid is of great medical relevance as it is found in several major incurable diseases, like Alzheimer's and Parkinson's disease and diabetes mellitus. Work on recombinant BRICHOS domains and transfected cells indicate that BRICHOS is a chaperone domain that, during biosynthesis, binds to precursor protein regions with high β-sheet propensities, thereby preventing them from amyloid formation. Regions prone to form β-sheets are present in all BRICHOS-containing precursor proteins and are probably eventually released by proteolytic cleavage, generating different peptides with largely unknown bioactivities. Recombinant BRICHOS domains from Bri2 and proSP-C have been found to efficiently prevent SP-C, the amyloid β-peptide associated with Alzheimer's disease, and medin, found in aortic amyloid, from forming amyloid fibrils. The data collected so far on BRICHOS raise several interesting topics for further research: (a) amyloid formation is a potential threat for many more proteins than the ones recognized so far in amyloid diseases; (b) amyloid formation of widely different peptides involves intermediate(s) that are recognized by the BRICHOS domain, suggesting that they have distinct structural similarities; and (c) the BRICHOS domain might be harnessed in therapeutic strategies against amyloid diseases.     

BRICHOS Domains Efficiently Delay Fibrillation of Amyloid β-Peptide

Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in β-sheet structure. The BRICHOS domain consisting of ～100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid β-peptides (Aβ(40) and Aβ(42)) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aβ is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded β-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended β-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.     

The activation mechanism of Hsp26 does not require dissociation of the oligomer

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.     

具有分子伴侣功能的抗体

蛋白质的折叠问题一直是生物学研究的前沿之一，蛋白质稳态平衡的破坏与衰老及很多神经退行性疾病的发病机理密切相关，而蛋白质的正确折叠与蛋白质稳态在很大程度上取决于分子伴侣参与构建的复杂网络。许多研究表明，抗体可以作为分子伴侣促进蛋白质的正确折叠，并阻止蛋白质的异常聚集，抗体所具有的严格底物特异性使其具备了治疗特定蛋白质折叠病、帮助包涵体复性等应用潜力。本文简要介绍了分子伴侣的研究进展，详细阐述了具有分子伴侣功能的抗体及单链抗体的研究进展，最后重点讨论了可抑制蛋白质聚集的抗体的研究近况。     

Protein disulfide isomerase

During the maturation of extracellular proteins, disulfide bonds that chemically cross-link specific cysteines are often added to stabilize a protein or to join it covalently to other proteins. Disulfide formation, which requires a change in the covalent structure of the protein, occurs as the protein folds into its three-dimensional structure. In the eukaryotic endoplasmic reticulum and in the bacterial periplasm, an elaborate system of chaperones and folding catalysts ensure that disulfides connect the proper cysteines and that the folding protein does not make improper interactions. This review focuses specifically on one of these folding assistants, protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide formation and isomerization and a chaperone that inhibits aggregation.     

The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the Escherichia coli Ribosome

Background

Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.

Results

We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.

Conclusion

The ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.     

Folding on the chaperone: yield enhancement through loose binding

A variety of small cageless chaperones have been discovered that can assist protein folding without the consumption of ATP. These include mini-chaperones (catalytically active fragments of larger chaperones), as well as small proteins such as alpha-casein and detergents acting as "artificial chaperones." These chaperones all possess exposed hydrophobic patches on their surface that act as recognition sites for misfolded proteins. They lack the complexity of chaperonins (that encapsulate proteins in their inner rings) and their study can offer insight into the minimal requirements for chaperone function. We use molecular dynamics simulations to investigate how a cageless chaperone, modeled as a sphere of tunable hydrophobicity, can assist folding of a substrate protein. We find that under steady-state (non-stress) conditions, cageless chaperones that bind to a single substrate protein increase folding yields by reducing the time the substrate spends in an aggregation-prone state in a dual manner: (a) by competing for aggregation-prone hydrophobic sites on the surface of a protein, hence reducing the time the protein spends unprotected in the bulk and (b) by accelerating folding rates of the protein. In both cases, the chaperone must bind to and hold the protein loosely enough to allow the protein to change its conformation and fold while bound. Loose binding may enable small cageless chaperones to help proteins fold and avoid aggregation under steady-state conditions, even at low concentrations, without the consumption of ATP.     

The endoplasmic reticulum HSP40 co‐chaperone ERdj3/DNAJB11 assembles and functions as a tetramer

ERdj3/DNAJB11 is an endoplasmic reticulum (ER)‐targeted HSP40 co‐chaperone that performs multifaceted functions involved in coordinating ER and extracellular proteostasis. Here, we show that ERdj3 assembles into a native tetramer that is distinct from the dimeric structure observed for other HSP40 co‐chaperones. An electron microscopy structural model of full‐length ERdj3 shows that these tetramers are arranged as a dimer of dimers formed by distinct inter‐subunit interactions involving ERdj3 domain II and domain III. Targeted deletion of residues 175‐190 within domain II renders ERdj3 a stable dimer that is folded and efficiently secreted from mammalian cells. This dimeric ERdj3 shows impaired substrate binding both in the ER and extracellular environments and reduced interactions with the ER HSP70 chaperone BiP. Furthermore, we show that overexpression of dimeric ERdj3 exacerbates ER stress‐dependent reductions in the secretion of a destabilized, aggregation‐prone protein and increases its accumulation as soluble oligomers in extracellular environments. These results reveal ERdj3 tetramerization as an important structural framework for ERdj3 functions involved in coordinating ER and extracellular proteostasis in the presence and absence of ER stress.     

Reducing ER stress with chaperone therapy reverses sleep fragmentation and cognitive decline in aged mice

As the aging population grows, the need to understand age‐related changes in health is vital. Two prominent behavioral changes that occur with age are disrupted sleep and impaired cognition. Sleep disruptions lead to perturbations in proteostasis and endoplasmic reticulum (ER) stress in mice. Further, consolidated sleep and protein synthesis are necessary for memory formation. With age, the molecular mechanisms that relieve cellular stress and ensure proper protein folding become less efficient. It is unclear if a causal relationship links proteostasis, sleep quality, and cognition in aging. Here, we used a mouse model of aging to determine if supplementing chaperone levels reduces ER stress and improves sleep quality and memory. We administered the chemical chaperone 4‐phenyl butyrate (PBA) to aged and young mice, and monitored sleep and cognitive behavior. We found that chaperone treatment consolidates sleep and wake, and improves learning in aged mice. These data correlate with reduced ER stress in the cortex and hippocampus of aged mice. Chaperone treatment increased p‐CREB, which is involved in memory formation and synaptic plasticity, in hippocampi of chaperone‐treated aged mice. Hippocampal overexpression of the endogenous chaperone, binding immunoglobulin protein (BiP), improved cognition, reduced ER stress, and increased p‐CREB in aged mice, suggesting that supplementing BiP levels are sufficient to restore some cognitive function. Together, these results indicate that restoring proteostasis improves sleep and cognition in a wild‐type mouse model of aging. The implications of these results could have an impact on the development of therapies to improve health span across the aging population.     

Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae

Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.     

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Beyond Transcription—New Mechanisms for the Regulation of Molecular Chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions—the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions.     

Beyond transcription--new mechanisms for the regulation of molecular chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions-the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T‐complex protein‐1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.