The Pharmacology and Dual Role of Proteostasis in Amyloidoses

Systemic amyloidosis is a relatively rare multisystem disease caused by the deposition of misfolded protein in various tissues and organs. It may present to almost any specialty, and diagnosis is frequently delayed.[1][1] Cardiac involvement is a leading cause of morbidity and mortality, especially

The Clinical Impact of Proteomics in Amyloid Typing

In addition to the sigma(32)-mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADP-liganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40 degrees C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.

Protein misfolding and aggregation is the phenomenon of the generic propensity of proteins, considered as a dark side of the protein world, and its exact mechanism is still not deciphered. Understanding the complexity of protein aggregation is currently the primary apprehension and challenge in biology and medicine due to their association with various debilitating human proteinopathies and neurodegenerative diseases. The mechanism of protein aggregation, associated diseases, and the development of efficient therapeutic strategies against these diseases are very challenging. These diseases are caused by different proteins, each protein with different mechanisms and consisting of various microscopic phases or events. These microscopic steps are functioning on different timescales during aggregation. Here, we highlighted the different features and current trends in protein aggregation. The study thoroughly recapitulates the various factors influencing, possible causes, types of aggregates and aggregation, their different proposed mechanisms, and the methods used to study the aggregation. Additionally, the formation and elimination of misfolded or aggregated proteins in the cell, the role of the ruggedness of the protein folding landscape in protein aggregation, proteinopathies, and the challenges for their prevention are comprehensively elucidated. A holistic understanding of different aspects of aggregation, molecular steps governing the various features of protein quality control, and crucial queries about the modulation of these processes and their interactions with other systems in cellular protein quality control can be considered conducive to comprehending the mechanism, designing effective approaches towards prevention of protein aggregation, rationalizing the etiology and development of novel strategies against therapy and management of the proteinopathies.

Protein aggregation is now a common hallmark of numerous human diseases, most of which involve cytosolic aggregates including Aβ (AD) and ⍺-synuclein (PD) in Alzheimer's disease and Parkinson's disease. However, it is also evident that protein aggregation can also occur in the lumen of the endoplasmic reticulum (ER) that leads to specific diseases due to loss of protein function or detrimental effects on the host cell, the former is inherited in a recessive manner where the latter are dominantly inherited. However, the mechanisms of protein aggregation, disaggregation and degradation in the ER are not well understood. Here we provide an overview of factors that cause protein aggregation in the ER and how the ER handles aggregated proteins. Protein aggregation in the ER can result from intrinsic properties of the protein (hydrophobic residues in the ER), oxidative stress or nutrient depletion. The ER has quality control mechanisms [chaperone functions, ER-associated protein degradation (ERAD) and autophagy] to ensure only correctly folded proteins exit the ER and enter the cis-Golgi compartment. Perturbation of protein folding in the ER activates the unfolded protein response (UPR) that evolved to increase ER protein folding capacity and efficiency and degrade misfolded proteins. Accumulation of misfolded proteins in the ER to a level that exceeds the ER-chaperone folding capacity is a major factor that exacerbates protein aggregation. The most significant ER resident protein that prevents protein aggregation in the ER is the heat shock protein 70 (HSP70) homologue, BiP/GRP78, which is a peptide-dependent ATPase that binds unfolded/misfolded proteins and releases them upon ATP binding. Since exogenous factors can also reduce protein misfolding and aggregation in the ER, such as chemical chaperones and antioxidants, these treatments have potential therapeutic benefit for ER protein aggregation-associated diseases.

A key pathological feature of neurodegenerative disorders such as Parkinson’s disease is the generation of misfolded and aggregated proteins. Cationic arginine-rich peptides (CARPs), which include poly-arginine peptides, have shown potent neuroprotective capabilities in various neurological injury models, and may have a potential therapeutic role in chronic neurodegenerative disorders. The present study sought to investigate whether the CARP, Poly-arginine-18 (R18), could prevent protein aggregation. Poly-arginine R18 (R18), along with L-arginine (Arg) were evaluated for interactions with the protein, lysozyme, using in silico molecular docking simulations. An in vitro heat-induced lysozyme aggregation assay was also established, and used to examine the ability of Arg and R18 to inhibit protein aggregation of the protein. In silico binding affinity studies found that Arg and R18 bound the C-terminal region of lysozyme via multiple hydrogen bonds, providing evidence for a direct protein-protein interaction. In addition, both Arg and R18 reduced heat-induced lysozyme aggregation in vitro, but R18 was more effective at lower concentrations. This study demonstrates that the R18 polyarginine peptide can bind to lysozyme and prevent aggregation through a direct protein interaction. Such functionality has potential implications for promoting protein solubility and, thus, treating disorders associated with abnormal protein aggregation, including synucleinopathies such as Parkinson’s disease.

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.

Protein homeostasis networks in physiology and disease

Aberrant protein aggregation is a defining feature of most neurodegenerative diseases. During pathological aggregation, key proteins transition from their native state to alternative conformations, which are prone to oligomerize into highly ordered fibrillar states. As part of the cellular quality control machinery, molecular chaperones can intervene at many stages of the aggregation process to inhibit or reverse aberrant protein aggregation or counteract the toxicity associated with amyloid species. Although the action of chaperones is considered cytoprotective, essential housekeeping functions can be hijacked for the propagation and spreading of protein aggregates, suggesting the cellular protein quality control system constitutes a double-edged sword in neurodegeneration. Here, we discuss the various mechanisms used by chaperones to influence protein aggregation into amyloid fibrils to understand how the interplay of these activities produces specific cellular outcomes and to define mechanisms that may be targeted by pharmacological agents for the treatment of neurodegenerative conditions.

Transfer RNA (tRNA) modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding. However, the mechanisms by which tRNA modifications modulate codon optimality and trigger phenotypes remain unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs in wobble uridine modification (U34) mutants. This triggers ribosome collisions and a coordinated hierarchical response of cellular quality control pathways. High-resolution ribosome profiling reveals an unexpected functional diversity of U34 modifications during decoding. For instance, 5-carbamoylmethyluridine (ncm5U) exhibits distinct effects at the A and P sites. Importantly, ribosomes only slow down at a fraction of codons decoded by hypomodified tRNA, and the decoding speed of most codons remains unaffected. However, the translation speed of a codon largely depends on the identity of A- and P-site codons. Stalling at modification-dependent codon pairs induces ribosome collisions, triggering ribosome-associated quality control (RQC) and preventing protein aggregation by degrading aberrant nascent peptides and messenger RNAs. Inactivation of RQC stimulates the expression of molecular chaperones that remove protein aggregates. Our results demonstrate that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs, showing the coordinated regulation and adaptability of cellular surveillance systems. These systems ensure efficient and accurate protein synthesis and maintain protein homeostasis.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Toxicity associated with abnormal protein folding and protein aggregation are major hypotheses for neurodegeneration. This article comparatively reviews the experimental and human tissue-based evidence for the involvement of such mechanisms in neuronal death associated with the motor system disorders of X-linked spinobulbar muscular atrophy (SBMA; Kennedy's disease) and amyotrophic lateral sclerosis (ALS), especially disease related to mutations in the superoxide dismutase (SOD1) gene. Evidence from transgenic mouse, Drosophila and cell culture models of SBMA, in common with other trinucleotide repeat expansion disorders, show protein aggregation of the mutated androgen receptor, and intraneuronal accumulation of aggregated protein, to be obligate mechanisms. Strong experimental data link these phenomena with downstream biochemical events involving gene transcription pathways (CREB-binding protein) and interactions with protein chaperone systems. Manipulations of these pathways are already established in experimental systems of trinucleotide repeat disorders as potential beneficial targets for therapeutic activity. In contrast, the evidence for the role of protein aggregation in models of SOD1-linked familial ALS is less clear-cut. Several classes of intraneuronal inclusion body have been described, some of which are invariably present. However, the lack of understanding of the biochemical basis of the most frequent inclusion in sporadic ALS, the ubiquitinated inclusion, has hampered research. The toxicity associated with expression of mutant SOD1 has been intensively studied however. Abnormal protein aggregation and folding is the only one of the four major hypotheses for the mechanism of neuronal degeneration in this disorder currently under investigation (the others comprise oxidative stress, axonal transport and cytoskeletal dysfunctions, and glutamatergic excitotoxicity). Whilst hyaline inclusions, which are strongly immunoreactive to SOD1, are linked to degeneration in SOD1 mutant mouse models, the evidence from human tissue is less consistent and convincing. A role for mutant SOD1 aggregation in the mitochondrial dysfunction associated with ALS, and in potentially toxic interactions with heat shock proteins, both leading to apoptosis, are supported by some experimental data. Direct in vitro data on mutant SOD1 show evidence for spontaneous oligomerization, but the role of such oligomers remains to be elucidated, and therapeutic strategies are less well developed for this familial variant of ALS.

Massive production and accumulation of a single abnormal protein may constitute a major toxic burden for the cell and even compromise the organism's long-term viability. Consequently, adaptation and survival have forced evolution to create 'quality control' mechanisms that detect, monitor, and often degrade such abnormally folded gene products, in which molecular chaperones are key players. Notwithstanding this, there are numerous examples of misfolded proteins which, in spite of being recognized as aberrant and efficiently discarded by cellular quality control, still retain some of the functional properties of their wild-type counterparts, so that their maintenance in the cell would be beneficial for the organism. Herein are described the cellular roles of molecular chaperones and some new insights on the mechanisms by which they influence the development of human diseases caused by mutations that lead to protein misfolding. A special emphasis is given to cystic fibrosis, a classical genetic disorder resulting from the retention and degradation of a mutant, albeit functional, protein by the endoplasmic reticulum quality control. This particular system has been a good example to describe the mechanisms that are likely to be shared by a number of protein substrates, to define the common characteristics of the mutants, as well as to identify the mechanistic intervenients in their retention and degradation. Finally, new approaches aimed at correcting protein folding defects are discussed, including the potential of molecular chaperones (e.g., through RNA interference) as novel therapeutic targets, and the usage of chemical or pharmacological chaperones as new therapeutic agents.

A 74-year-old man presented with decreasing exercise tolerance and mild ankle edema. He was previously fit but was now breathless on climbing 2 flights of stairs. He had no history of angina, orthopnea, or paroxysmal nocturnal dyspnea. His medical history included non–insulin-dependent diabetes mellitus treated for 10 years and mild hypertension. Six years earlier he had been diagnosed with a monoclonal gammopathy of unknown significance. At that time, a bone marrow biopsy showed 30% overall cellularity with 5% to 10% plasmacytosis (normal <4%) and immunoglobulin light-chain restriction. Approximately 3 years ago, he developed deep vein thrombosis and was treated with low-molecular-weight heparin. A year later, leg swelling occurred and was attributed to venous insufficiency. The following year, he developed progressive fatigue on exertion, and an abnormal ECG (Figure 1) led to a treadmill test that was considered normal. An echocardiogram showed concentric wall thickening (Movie 1 in the Data Supplement), and the possibility of cardiac amyloidosis was raised. A fat pad biopsy was negative for amyloid deposits. The bone marrow biopsy performed in 2005 (when his monoclonal gammopathy of unknown significance was diagnosed) was restained and was negative for amyloid. At that time, serum-free λ light chains were 108.9 mg/L (normal range, 5.7–26.3) with κ light chains of 13 mg/L (normal, 3.3–19) and an abnormal ratio of 0.12 (normal, 0.26–1.65). His brain natriuretic peptide measured 275 pcg/mL. He was treated with oral diuretics, which improved leg swelling, but because of persistent symptoms, he sought medical care at our institution. On review of symptoms, he denied jaw claudication, symptoms of postural hypotension, easy bruising, or tongue swelling. He did give a history suggestive of neuropathy with a leathery feeling in his feet but no numbness in his hands. Medications included metformin 500 mg twice a day, aspirin 80 mg daily, lisinopril …

Special series: Molecular chaperones in protein folding and disease