Reverse Protein Aggregation Research Articles

Decoding Skd3 (Human CLPB): a Mitochondrial Protein Disaggregase Critical for Human Health

AbstractProtein folding is important for all life. Indeed, protein misfolding can result in catastrophic protein aggregation and toxicity. The pathways involved in reversing protein aggregation within human mitochondria had long been unknown. We recently discovered that Skd3 (human CLPB) is a potent mitochondrial protein disaggregase, which is regulated by the rhomboid protease PARL, and maintains the solubility of many important mitochondrial proteins. Skd3 variants underlie several debilitating human diseases, including 3‐methylglutaconic aciduria, severe congenital neutropenia, and premature ovarian insufficiency. Here, we describe advances in understanding Skd3 function, mechanism, and structure and place these discoveries in the context of physiology and disease.

Proteostasis and Ribostasis Impairment as Common Cell Death Mechanisms in Neurodegenerative Diseases.

The cellular homeostasis of proteins (proteostasis) and RNA metabolism (ribostasis) are essential for maintaining both the structure and function of the brain. However, aging, cellular stress conditions, and genetic contributions cause disturbances in proteostasis and ribostasis that lead to protein misfolding, insoluble aggregate deposition, and abnormal ribonucleoprotein granule dynamics. In addition to neurons being primarily postmitotic, nondividing cells, they are more susceptible to the persistent accumulation of abnormal aggregates. Indeed, defects associated with the failure to maintain proteostasis and ribostasis are common pathogenic components of age-related neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, the neuronal deposition of misfolded and aggregated proteins can cause both increased toxicity and impaired physiological function, which lead to neuronal dysfunction and cell death. There is recent evidence that irreversible liquid-liquid phase separation (LLPS) is responsible for the pathogenic aggregate formation of disease-related proteins, including tau, α-synuclein, and RNA-binding proteins, including transactive response DNA-binding protein 43, fused in sarcoma, and heterogeneous nuclear ribonucleoprotein A1. Investigations of LLPS and its control therefore suggest that chaperone/disaggregase, which reverse protein aggregation, are valuable therapeutic targets for effective treatments for neurological diseases. Here we review and discuss recent studies to highlight the importance of understanding the common cell death mechanisms of proteostasis and ribostasis in neurodegenerative diseases.

Amphiphilic dendrons as supramolecular holdase chaperones.

The aggregation of incompletely or incorrectly folded proteins is implicated in diseases including Alzheimer's, cataracts, and other maladies. Natural systems express protein chaperones to prevent or even reverse harmful protein aggregation. Synthetic chaperone-like systems have sought to mimic the action of their biological counterparts but typically require substantial optimization and high concentrations to be functional, or lack programmability that would enable the targeting of specific protein substrates. Here we report a series of amphiphilic dendrons that undergo assembly and inhibit the aggregation of fragment 16-22 amyloid β protein (Aβ16-22). We show that monodisperse dendrons with hydrophilic tetraethylene glycol chains and a hydrophobic core based on naphthyl and benzyl ethers undergo supramolecular assembly in aqueous solutions to form sphere-like particles. The solubility of these dendrons and their assemblies is tuned by varying the relative sizes of their hydrophilic and hydrophobic regions. Two water-soluble dendrons are discovered and shown, via fluorescence experiments with rhodamine 6G, to generate a hydrophobic environment. Furthermore, we demonstrate that sub-stoichiometric concentrations of these amphiphilic dendrons stabilize Aβ16-22 peptide with respect to aggregation, mimicking the activity of holdase chaperones. Our results highlight the potential of these amphiphilic molecules as the basis for a novel approach to artificial chaperones that may address many of the challenges associated with existing synthetic chaperone mimics.

Developing therapeutic protein disaggregases for Neurodegenerative Disease

The accumulation of abnormal protein aggregates in human brain is connected with neurodegenerative diseases. The mammalian protein‐disaggregase system comprised of Hsp110, Hsp70, and Hsp40 can protect cells from proteinopathies. However, this disaggregase system has limitations and its activity declines with age. As a therapeutic strategy for neurodegenerative disease we propose to introduce an exogenous, synthetic disaggregase machinery, based on Hsp104 that converts amyloid to soluble, functional protein. Hsp104 is a hexameric AAA+ ATPase that is critical for stress tolerance in yeast by facilitating the resolubilization of stress‐damaged, aggregated proteins. The homologues of Hsp104 are highly conserved in bacteria, fungi, and plants, but absent from metazoa. The disaggregase activity of Hsp104 can be enhanced by the presence Hsp70 and Hsp40. Hsp104 can dissolve α‐synuclein, β‐amyloid, and TDP‐43 aggregates, but impractically high Hsp104 concentrations are needed. We aim to rationally design potentiated Hsp104 variants that can reverse protein misfolding at low concentrations. One potential problem for the currently discovered potentiated Hsp104 variants is their “off‐target” effects, where expression potentiated Hsp104 variant in ∆hsp104 yeast causes cell death possibly by unfolding metastable proteins at 37°C. By investigating the mechanism of Hsp104 disaggregation and its structural basis, we will engineer and evolve potentiated Hsp104 variants with minimized “off‐target” effect that eradicate α‐synuclein, β‐amyloid, or TDP‐43 misfolding and toxicity. We have exploited new cryo‐EM structures of Hsp104 to engineer key interfaces to yield several novel Hsp104 variants with tunable activity, which can reverse protein aggregation in the presence or absence of human Hsp70 and Hsp40. These novel Hsp104 variants effectively rescue the aggregation and toxicity of diverse human neurodegenerative disease proteins and could have therapeutic utility. Moreover, our engineered Hsp104 variants provide key mechanistic insights into disaggregase function.

Steroid-Quinoline Hybrids for Disruption and Reversion of Protein Aggregation Processes.

Reversing protein aggregation within cells may be an important tool to fight protein-misfolding disorders such as Alzheimer's, Parkinson's, and cardiovascular diseases. Here we report the design and synthesis of a family of steroid-quinoline hybrid compounds based on the framework combination approach. This set of hybrid compounds effectively inhibited Aβ1-42 self-aggregation in vitro by delaying the exponential growth phase and/or reducing the quantity of fibrils in the steady state. Their disaggregation efficacy was further demonstrated against preaggregated Aβ1-42 peptides in cellular assays upon their endocytosis by neuroblastoma cells, as they reverted both the number and the average area of fibrils back to basal levels. The antiaggregation effect of these hybrids was further tested and demonstrated in a cellular model of general protein aggregation expressing a protein aggregation fluorescent sensor. Together, our results show that the new cholesterol-quinoline hybrids possess wide and marked disaggregation capacities and are therefore promising templates for the development of new drugs to deal with conformational disorders.

Yeast Proteins may Reversibly Aggregate like Amphiphilic Molecules

More than a hundred proteins in yeast reversibly aggregate and phase-separate in response to various stressors, such as nutrient depletion and heat shock. We know little about the protein sequence and structural features behind this ability, which has not been characterized on a proteome-wide level. To identify the distinctive features of aggregation-prone protein regions, we apply machine learning algorithms to genome-scale limited proteolysis-mass spectrometry (LiP-MS) data from yeast proteins. LiP-MS data reveals that 96 proteins show significant structural changes upon heat shock. We find that in these proteins the propensity to phase separate cannot be solely driven by disordered regions, because their aggregation-prone regions (APRs) are not significantly disordered. Instead, the phase separation of these proteins requires contributions from both disordered and structured regions. APRs are significantly enriched in aliphatic residues and depleted in positively charged amino acids. Aggregator proteins with longer APRs show a greater propensity to aggregate, a relationship that can be explained by equilibrium statistical thermodynamics. Altogether, our observations suggest that proteome-wide reversible protein aggregation is mediated by sequence-encoded properties. We propose that aggregating proteins resemble supra-molecular amphiphiles, where APRs are the hydrophobic parts, and non-APRs are the hydrophilic parts.

Developing therapeutic protein disaggregases for neurodegenerative disease

AbstractBackgroundThe accumulation of abnormal protein aggregates in human brain is connected with neurodegenerative diseases. The mammalian protein‐disaggregase system comprised of Hsp110, Hsp70, and Hsp40 can protect cells from proteinopathies. However, this disaggregase system has limitations and its activity declines with age. As a therapeutic strategy for neurodegenerative disease we propose to introduce an exogenous, synthetic disaggregase machinery, based on Hsp104 that converts amyloid to soluble, functional protein. Hsp104 is a hexameric AAA+ ATPase that is critical for stress tolerance in yeast by facilitating the resolubilization of stress‐damaged, aggregated proteins. The homologues of Hsp104 are highly conserved in bacteria, fungi, and plants, but absent from metazoa. The disaggregase activity of Hsp104 can be enhanced by the presence Hsp70 and Hsp40. Hsp104 can dissolve α‐synuclein, β‐amyloid, and TDP‐43 aggregates, but impractically high Hsp104 concentrations are needed.MethodNimble yeast models are used to perform an initial evaluation to enhance experimental efficiency. Promising variants will be purified for further characterization and will be advanced to neuronal and glial cell models.ResultWe have engineered and evolved potentiated Hsp104 variants with minimized “off‐target” effect that eradicate α‐synuclein, β‐amyloid, or TDP‐43 misfolding and toxicity. We have discovered Hsp104 variant that reverse TDP‐43 toxicity specifically with no "off‐target" effect. We have exploited new cryo‐EM structures of Hsp104 to engineer key interfaces to yield several novel Hsp104 variants with tunable activity, which can reverse protein aggregation in the presence or absence of human Hsp70 and Hsp40.ConclusionThese novel Hsp104 variants effectively rescue the aggregation and toxicity of diverse human neurodegenerative disease proteins and could have therapeutic utility.

Acute Heat Stress Leads to Reversible Aggregation of Nuclear Proteins into Nucleolar Rings in Fission Yeast

Upon acute heat stress (HS), overall mRNA transcription, processing, and export are inhibited, leading to cell growth arrest. However, how cells turn off mRNA metabolism is not fully understood. Here, we show that acute HS results in the segregation and aggregation of multiple nuclear and nucleolar proteins into ring-like structures located at the nucleolar periphery (nucleolar rings [NuRs]). NuRs sequester essential factors required for nuclear mRNA metabolism and nuclear pore complex function, as well as cell-cycle regulators. When cells are switched back to growing temperatures, NuRs disaggregate, and their components relocate to their functional environments in an Hsf1- and Hsp104-dependent manner, and concomitantly with the reinitiation of cell growth. These findings highlight the contribution of reversible protein aggregation to the inhibition of overall RNA-related activities in the nucleus and its functional relevance in the maintenance of cellular homeostasis during acute HS.

Processive extrusion of polypeptide loops by a Hsp100 disaggregase.

The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14.

A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer's Disease.

Age-progressive neurodegenerative pathologies, including Alzheimer’s disease (AD), are distinguished and diagnosed by disease-specific components of intra- or extra-cellular aggregates. Increasing evidence suggests that neuroinflammation promotes protein aggregation, and is involved in the etiology of neurological diseases. We synthesized and tested analogs of the naturally occurring tubulin-binding compound, combretastatin A-4. One such analog, PNR502, markedly reduced the quantity of Alzheimer-associated amyloid aggregates in the BRI-Aβ1–42 mouse model of AD, while blunting the ability of the pro-inflammatory cytokine IL-1β to raise levels of amyloid plaque and its protein precursors in a neuronal cell-culture model. In transgenic Caenorhabditis elegans (C. elegans) strains that express human Aβ1–42 in muscle or neurons, PNR502 rescued Aβ-induced disruption of motility (3.8-fold, P < 0.0001) or chemotaxis (1.8-fold, P < 0.05), respectively. Moreover, in C. elegans with neuronal expression of Aβ1–42, a single day of PNR502 exposure reverses the chemotaxis deficit by 54% (P < 0.01), actually exceeding the protection from longer exposure. Moreover, continuous PNR502 treatment extends nematode lifespan 23% (P ≤ 0.001). Given that PNR502 can slow, prevent, or reverse Alzheimer-like protein aggregation in human-cell-culture and animal models, and that its principal predicted and observed binding targets are proteins previously implicated in Alzheimer’s, we propose that PNR502 has therapeutic potential to inhibit cerebral Aβ1–42 aggregation and prevent or reverse neurodegeneration.

Protein aggregation in bacteria.

ABSTRACTProtein aggregation occurs as a consequence of perturbations in protein homeostasis that can be triggered by environmental and cellular stresses. The accumulation of protein aggregates has been associated with aging and other pathologies in eukaryotes, and in bacteria with changes in growth rate, stress resistance and virulence. Numerous past studies, mostly performed in Escherichia coli, have led to a detailed understanding of the functions of the bacterial protein quality control machinery in preventing and reversing protein aggregation. However, more recent research points toward unexpected diversity in how phylogenetically different bacteria utilize components of this machinery to cope with protein aggregation. Furthermore, how persistent protein aggregates localize and are passed on to progeny during cell division and how their presence impacts reproduction and the fitness of bacterial populations remains a controversial field of research. Finally, although protein aggregation is generally seen as a symptom of stress, recent work suggests that aggregation of specific proteins under certain conditions can regulate gene expression and cellular resource allocation. This review discusses recent advances in understanding the consequences of protein aggregation and how this process is dealt with in bacteria, with focus on highlighting the differences and similarities observed between phylogenetically different groups of bacteria.

Revisiting the earliest signatures of amyloidogenesis: Roadmaps emerging from computational modeling and experiment

Neurodegenerative amyloidogenesis begins with the aggregation of intrinsically disordered proteins (IDPs), which is the first step in a cascade of assembly events that can lead to insoluble fibrous deposits in brain tissue. IDP conformations that promote formation of toxic oligomers remain poorly understood, and are the most fundamental target of putative treatments for neurodegenerative disease. Rapid advances in theory, simulation and experimental methods, hold the promise of reversing protein aggregation by identifying and developing inhibitors of the transient amyloidogenic IDP conformations. To make meaningful progress it is important to appreciate the benefits and limitations of the latest developments in computational methods of conformational and ensemble modeling, and their integration and validation with experiments. Integrated studies are beginning to provide significant conceptual and mechanistic insights, including identification of the properties of amyloidogenic IDPs in their free, unbound form. At the same time, contradicting viewpoints have emerged concerning convergence of IDP ensemble signatures and properties from parallel studies, and there also remains a pressing need to develop physical models that can deliver reliable predictions across different IDP families. Focussing on the four most common amyloidogenic IDPs of Amyloid β, Tau, α‐synuclein and Prions, improvements are proposed for next‐generation models and experiments that can potentially identify drug treatments for neurodegenerative disease via incorporation of the extended cellular environment.This article is categorized under: Molecular and Statistical Mechanics &gt; Molecular Mechanics Structure and Mechanism &gt; Computational Biochemistry and Biophysics Structure and Mechanism &gt; Molecular Structures

Toxoplasma gondii Clp family protein: TgClpB1 plays a crucial role in thermotolerance.

Caseinolytic peptidase B (ClpB) plays a pivotal role in suppressing and reversing protein aggregation. Toxoplasma gondii is an intracellular parasitic protozoan that infects a wide variety of mammals and birds and therefore is exposed to a broad range of living condition. We screened ToxoDB (http://ToxoDB.org) and identified 10 putative T. gondii genes encoding members of the Clp superfamily of caseinolytic proteases and chaperones. Of these, we focused on characterizing the Class I ATP-dependent molecular chaperones TgClpB1, TgClpB2, and TgClpB3. We found that TgClpB1, the most divergent of the five T. gondii Class I Clp ATPases, is cytoplasmic, TgClpB2 is found in the mitochondria of the parasites, and TgClpB3 is a ClpB with novel apicoplast localization. Knockout strains of TgClpB1 and TgClpB2 were established by CRISPR/Cas9 mutagenesis, and their complementing strains were constructed with FLAG-tag. Although knockout of TgClpB1 or TgClpB2 did not affect growth under normal circumstances, TgClpB1 was required for T. gondii thermotolerance. The growth, replication, and invasion capabilities of TgClpB1-deficient mutants were significantly inhibited after extracellular parasites were pretreated at 45°C. Moreover, TgClpB1 were observed at the poles of the ΔTgClpB1 FLAG-tagged strain treated at 42°C.

Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress.

Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.

Protein-Remodeling Factors As Potential Therapeutics for Neurodegenerative Disease.

Protein misfolding is implicated in numerous neurodegenerative disorders including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. A unifying feature of patients with these disorders is the accumulation of deposits comprised of misfolded protein. Aberrant protein folding can cause toxicity through a loss or gain of protein function, or both. An intriguing therapeutic approach to counter these disorders is the application of protein-remodeling factors to resolve these misfolded conformers and return the proteins to their native fold and function. Here, we describe the application of protein-remodeling factors to alleviate protein misfolding in neurodegenerative disease. We focus on Hsp104, Hsp110/Hsp70/Hsp40, NMNAT, and HtrA1, which can prevent and reverse protein aggregation. While many of these protein-remodeling systems are highly promising, their activity can be limited. Thus, engineering protein-remodeling factors to enhance their activity could be therapeutically valuable. Indeed, engineered Hsp104 variants suppress neurodegeneration in animal models, which opens the way to novel therapeutics and mechanistic probes to help understand neurodegenerative disease.

Effect of fat content and temperature on the translucency of Cheddar cheese

Cheese translucency is influenced by fat content, ripening and temperature and is traditionally determined by CIELAB whiteness. However, translucency is also affected by thickness of materials. The Kubelka-Munk method (K/S) estimates the ratio of absorbance to scatter from whiteness measurements on thin layers of materials above white and black backgrounds. The effect of fat content and temperature on Cheddar cheese translucency was evaluated using K/S and CIELAB methods. Cheddar cheeses varying in fat composition were made and translucency was measured during heating/cooling at 2 and 180 d of ripening. Low fat content in cheese was found to be related to high translucency at <30 °C. At 40 °C, translucency increased due to fat melting. At >60 °C, a reversible protein aggregation led to a reduction of translucency. Translucency increased as cheeses aged. CIELAB and K/S methods were highly correlated; K/S could therefore be useful for estimating cheese translucency.

DRUG DISCOVERY. A new dawn for cataracts.

Sterols reverse protein aggregation in an eye lens paradigm [Also see Report by Makley et al. ]

Feasibility of β-sheet breaker peptide-H102 treatment for Alzheimer's disease based on β-amyloid hypothesis.

β-amyloid hypothesis is the predominant hypothesis in the study of pathogenesis of Alzheimer's disease. This hypothesis claims that aggregation and neurotoxic effects of amyloid β (Aβ) is the common pathway in a variety of etiological factors for Alzheimer's disease. Aβ peptide derives from amyloid precursor protein (APP). β-sheet breaker peptides can directly prevent and reverse protein misfolding and aggregation in conformational disorders. Based on the stereochemical structure of Aβ1-42 and aggregation character, we had designed a series of β-sheet breaker peptides in our previous work and screened out a 10-residue peptide β-sheet breaker peptide, H102. We evaluated the effects of H102 on expression of P-tau, several associated proteins, inflammatory factors and apoptosis factors, and examined the cognitive ability of APP transgenic mice by behavioral test. This study aims to validate the β-amyloid hypothesis and provide an experimental evidence for the feasibility of H102 treatment for Alzheimer's disease.

In Vivo Disassembly and Reassembly of Protein Aggregates in Escherichia coli

Protein misfolding and aggregation are inevitable but detrimental cellular processes. Cells therefore possess protein quality control mechanisms based on chaperones and proteases that (re)fold or hydrolyze unfolded, misfolded, and aggregated proteins. Besides these conserved quality control mechanisms, the spatial organization of protein aggregates (PAs) inside the cell has been proposed as an important additional strategy to deal with their cytotoxicity. In the bacterium Escherichia coli, however, it remained unclear how this spatial organization is established and how this process of assembling PAs in the cell poles affects cellular physiology. In this report, high hydrostatic pressure was used to transiently reverse protein aggregation in living E. coli cells, allowing the subsequent (re)assembly of PAs to be studied in detail. This approach revealed PA assembly to be dependent on intracellular energy and metabolic activity, with the resulting PA structure being confined to the cell pole by nucleoid occlusion. Moreover, a correlation could be observed between the time needed for PA reassembly and the individual lag time of the cells, which might prevent symmetric segregation of cytotoxic PAs among siblings to occur and ensure rapid spatial clearance of molecular damage throughout the emerging population.

Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination

In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500–1,000 μmol photons m-2 s-1, and decreased at very high light intensity (1,500 μmol photons m-2 s-1). The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II.