This is Part 3 of a 5-part series. See also Part 1, Part 2, Part 4, and Part 5.

Topic 2: Protein Folding and Degradation
26 December 2007. In Alzheimer’s and related neurodegenerative diseases, misfolded proteins that normally are degraded instead accumulate and form aggregates. Therefore, understanding how neurons selectively destroy misfolded proteins can offer insight into the pathophysiology of AD. This section of the Bar Harbor 2007 report summarizes recent progress and points out major gaps in present-day understanding of how defects in protein folding and degradation might contribute to neurodegeneration.

Several different sensing and degradation systems for misfolded proteins exist in the human brain. Most are not well characterized, and few are known to respond strongly to an accumulation of toxic oligomeric proteins in neurodegenerative diseases. The ubiquitin proteasome system is one such system (see more detail below). It generally is not functioning as an effective protective response in these diseases, even though mutant forms of tau protein can become ubiquitinated and directed toward the proteasome in models of tauopathy. Numerous serum proteases degrade extracellular Aβ, but only the UPA-TPA system is induced by its oligomeric forms and can degrade it; other serum proteases clear primarily monomeric Aβ. Inside neurons, the ER-associated system of chaperones and proteasome-targeting factors (ERAD) does become activated in AD, as does the autophagy/lysosomal system. On the other hand, cytosolic chaperones that are part of the heat shock response do not. Additional intracellular proteolytic systems exist whose normal function and possible role in neurodegeneration are poorly understood. They include the calpains, the caspases, and cytosolic peptidases that work downstream of the proteasome. As a general rule, cytosolic and nuclear degradation systems are insufficiently upregulated in human neurodegenerative disease.

Another general theme emerged at the workshop. It is becoming accepted that a given protein does not exist merely in one structure, the native state, but assumes many different structures, some of which have biological functions and different interactions with degradation systems. As a rule, partly folded structures tend to penetrate membranes and unfolded structures get degraded. Biological processes regulate those states. There are equilibria between them, but the rate constants and fundamental kinetics and thermodynamics of the transitions are unknown. It is known that small changes in concentration of a given state can profoundly change its solution state and deposition. Some workshop participants felt that the field had accumulated enough data to begin quantitative modeling of the transitions.

Basic biochemistry research, particularly in bacteria, has built a body of knowledge in the past 30 years that established rapid degradation as the cell’s means to remove abnormal proteins of different sorts, including incomplete proteins, proteins resulting from missense mutations, free subunits of multimeric complexes, and oxidatively damaged proteins. Much of this degradation occurs in the ubiquitin proteasome, a soluble proteolytic system whose discovery and basic biology won the 2004 Nobel Prize in Chemistry. The proteasome differs fundamentally from other cellular proteases in that it depends on ATP for its function. Many neurodegenerative diseases feature pathologic inclusions of abnormal proteins with covalently linked ubiquitin, and even proteasomes associated with them. Ubiquitination of a target protein to mark it for degradation in the proteasome requires the sequential action of four types of enzyme, E1 through E4. Specificity arises partly from ubiquitin-carrying E2 proteins, but more so from the great variety of E3 ubiquitin ligases. (CHIP is an E3 of note in AD—it targets misfolded proteins attached to molecular chaperones, presumably as a fallback option after proper folding by CHIP has failed.)

The core 26S proteasome comprises a 20S core cylinder with dense walls that allow no lateral entry of ubiquitinated protein, and a less well-understood 19S component. Inside the cylinder, three different active sites (chymotrypsin-like, trypsin-like, caspase-like) cleave target proteins into peptides two to 24 amino acids long. Beyond their known role in antigen presentation, these scraps are understudied and may be important to neurodegenerative disease. The 19S complex consists of six ATPases and controls entry into the core cylinder. The ATPases may hold clues to neurodegenerative diseases, and their characterization in mammals requires much more study. This much is known, partly from research of homologous structures in Archae bacteria: the ATPases restrict entry of ubiquitinated target proteins into the 20S core. They act both to open the gate of the proteasome so the ubiquitin conjugate can bind, and to unfold the marked protein so it can be injected into the core. In this sense, the 19S complex is a chaperone. The ATPases form a ring on top of the proteasome, and their C-termini can align to fit into proteasome pockets to allow the gate to open. A conserved tyrosine residue is needed for this key-in-lock mechanism (Smith et al., 2007). The structural determinants and dynamics of this interaction should be studied further to explore the therapeutic potential of modulating gate-opening with small molecules designed to enhance protein degradation in neurodegenerative disease (Horwitz et al., 2007).

On the flip side, proteasome inhibitors are approved for the treatment of various forms of cancer. Some 50,000 cancer patients have undergone treatment with bortezomib to date, the first in this new class of drugs. The development of this drug was accompanied by questions about potential risks of neurodegenerative protein accumulation. This drug generally does not enter the brain, but one side effect that limits its application is a peripheral neuropathy with characteristic protein inclusions. Its effectiveness against cancer rests the twin findings that cancer cells are dependent on NFkB, whose activation requires proteasome degradation of IkB, and that cancer cells produce large numbers of misfolded proteins, which activate the unfolded protein response and cell death when the proteasome fails to degrade them.

Various hypotheses link proteasome dysfunction to neurodegenerative disease. They invoke, for example, binding of the proteasome to inclusions, rapid aggregation prior to degradation, or stopping up of the proteasome. None have been conclusively proven to date. One separate line of investigation has pointed to a new player in neurodegeneration. Surprisingly, the three active sites inside eukaryotic proteasomes are unable to cleave either normal or pathologically expanded polyQ sequences. The proteasome degrades most of a polyQ-containing protein; however, it ejects the polyQ tract undigested back into the cytosol, where those polyQ peptides either aggregate or become subject to degradation by a cytoplasmic enzyme. New research suggests that among all cytoplasmic proteases, only one is able to digest polyQ fragments. It is puromycin-sensitive aminopeptidase (PSA), a largely obscure enzyme (Bhutani et al., 2007). PSA is upregulated in polyQ disease and has drawn attention for its expression pattern in human brain areas resistant to tauopathy and for its ability to degrade tau and protect against neurodegeneration in a fly model of tauopathy (see ARF related news story; Sengupta et al., 2006; Karsten et al., 2006). Typically, fragments leaving the proteasome are destroyed within seconds. By contrast, PSA is a slow and inefficient way of degrading aggregation-prone or potentially toxic proteins. (Cathepsins in the lysosomal/autophagy pathway are able to degrade polyQ fragments, as well.)

This meeting session continued a prior discussion of protein misfolding in neurodegenerative disease. Besides Alzheimer’s, some 20 diseases of protein deposition are known, each of them characterized by a given protein that forms cross-β fibrils and other associated pathogenic intermediates. They are unrelated by sequence or native fold, raising the question of what it is that makes this particular set of proteins aggregate and cause disease. Recent research into the characteristics of aggregation showed that many more than those 20 disease proteins form amyloid fibrils as a default structure if given enough time. Indeed, the amyloid structure was found to be a generic polymer that is determined by the intrinsic properties of the polypeptide backbone. By contrast, the highly varied native structure of proteins is determined by the primary sequence and specific packing of amino acid side chains under regulated conditions (Fandrich and Dobson, 2002; Auer and Dobson, in press).

Like the ability to form fibrils, the toxicity of amyloid aggregates is also a generic feature. Amyloid aggregates from proteins that do not cause human disease, e.g., SH3 domains, can penetrate cells and cause similar toxicity as seen with known disease proteins. Furthermore, early aggregates, i.e., oligomers, of such generic proteins show the highest toxicity of the range of different species generated during aggregation, suggesting that oligomeric toxicity, too, is intrinsic (Baglioni et al., 2006). The reason why early forms of non-disease protein aggregates are most toxic is under intense investigation, but appears to lie partly with an increased exposure of hydrophobic surface while oligomers grow and convert to structures with a cross-β core (Cheon et al., 2007).

By contrast, while the ability of proteins to form fibrils is generic, their relative propensity to do so is not. Propensity varies dramatically with thermodynamic and kinetic parameters of a given context. In vitro, algorithms exist that can predict the intrinsic aggregation propensity of a given protein based on physicochemical principles such as charge and hydrophobicity (Chiti et al., 2003; Pawar et al., 2005). In vivo, the interconversions between different states of a protein underlie active control by molecular chaperones working in concert with quality control and degradation mechanisms. One hypothesis holds that when these protections fail, the proteins causing amyloid diseases revert to the stable, generic amyloid fibril and in the process generate a variety of toxic species.

A recent finding in this regard is that in vivo, proteins tend to occur in the cell at concentrations that put them close to the limit of their solubility. This arose from experiments plotting in-vitro aggregation rates of human proteins against their in-vivo concentrations derived from mRNA levels measured in tissue, which fell onto a near-perfect correlation of 0.97. This implies that even a subtle increase in concentration could drive up a given protein’s propensity to aggregate. In this way, small changes in protein concentration, in aggregation propensity, in quality control, or environmental factors could combine with longevity to cause disease (Tartaglia et al., 2007). This hypothesis is currently undergoing testing in fruit flies that express different forms of Aβ42 in brain and show amyloid deposition, movement deficits, and curtailed lifespan. Prior in-vitro work enables prediction of how specific mutations will affect the physicochemical properties and hence the intrinsic aggregation propensity of the resulting Aβ peptide. Mutations designed to either slow or speed up the aggregation rate generated fly strains whose histology, movement deficit, and survival improved or worsened accordingly. Even small changes in aggregation rate affected the flies’ survival drastically, and a given mutation’s propensity to form oligomers along the way correlated most strongly with survival (Luheshi et al., 2007). This system could be used for drug screening, as the ability to generate and test large numbers of flies renders statistically robust data.

Broadening research beyond Aβ, approaches at the systems biology level hold promise in addressing how the cell detects and responds to misfolded proteins globally. Systems biology can ask how protein homeostasis changes with age, and how it responds to the chronic stress of having a pathogenic, aggregation-prone species in the mix. These include proteins implicated in neurodegenerative diseases as well as a larger group of some 200 known diseases of protein conformation. There is consensus around the notion that these proteins adopt multiple states, some of which are toxic, and that the toxic states over time impair common pathways of cell function—folding, translocation between compartments, nuclear import-export, gene expression, etc. That is why studies trying to identify the toxicity mechanism of a given neurodegenerative disease protein have implicated virtually all aspects of cell function without converging around any one proposed mechanism. It is also widely agreed that cells have evolved the unfolded protein response (UPR) and the heat shock response to cope with the flow of aberrant proteins through the ER and cytoplasm, respectively. One open question in this area is why neurons in these diseases tend to activate the former but not the latter to remove toxic proteins from the cytoplasm.

Systems biology approaches to address these questions are feasible using genetics in model organisms. For example, recent studies of polyglutamine repeat disease in the C. elegans worm model have pointed to a group of some 350 genes that together form a “protein quality control proteome.” These genes can either enhance or suppress polyQ aggregation toxicity, and SOD1 aggregation toxicity, through effects on global mechanisms of protein homeostasis, or proteostasis. The current hypothesis holds that these genes make up a network of chaperones and clearance machines that maintain proteostasis. During normal biology, they support proper folding of polymorphic, mild folding variants, and clear misfolded forms. But the chronic presence of a mutant, highly aggregation-prone protein overwhelms the capacity of the proteostasis network such that even mild folding variants end up misfolding. This would lead not only to sequestration and loss of function of a variety of proteins from essential cellular processes, but also to toxic gains of function from the misfolded and accumulating species (Morimoto, 2006).

Supporting data for this hypothesis have come from studies using temperature-sensitive (ts) mutants as “folding sensors” to monitor protein homeostasis in C. elegans. Ts mutations are essentially mild folding mutations; prior developmental biology research has made many well-characterized examples available for study. The function, or absence of function, of a given ts protein reflects a fragile balance between its folding and degradation, making ts proteins suitable reporters of the global folding environment in the cell. Experiments coexpressing a given ts mutation with a polyQ protein at the threshold length of causing symptoms in human disease (i.e., Q40) showed that the presence of Q40 tracts abolished function of the ts mutant protein at the normally permissible temperature and killed the worms. Vice versa, the added presence of a ts mutation also rendered the Q40 protein more toxic. Ts mutations of many different proteins behaved in this way, showing that the enhanced misfolding is specific neither to cell type nor protein. In essence, the polyQ protein exposed the folding vulnerability of the ts protein, suggesting that it is competing for other proteins that are essential to maintain proteostasis. The presence of Q40 in the cell pushed the cell against the limit of its proteostasis capacity, presumably exhausting its ability to absorb additional misfolded proteins. Otherwise a single ts mutation could not markedly enhance polyQ toxicity (Gidalevitz, 2006).

The components and modifiers of a proteostatic network remain to be fully elucidated. One component is known modifiers of lifespan, i.e., genes in the insulin signaling pathway, such as daf-16. In genetic experiments, they both enhance proteostasis and suppress polyQ aggregation toxicity. (Insulin signaling is increasingly being implicated in AD, as well.) Heat shock factor-1, the upstream regulator of chaperones, is essential for this effect of the insulin signaling pathway (Morley et al., 2002; Morley et al., 2004; Hsu et al., 2003). Taken together, data from several labs suggest that Hsf-1, i.e., the classic stress response, does not merely protect the cell from acute damage but is critical for day-to-day protein homeostasis.

By contrast, aging works in the opposite direction in C. elegans. The ts mutations alone, without coexpressed Q40, aggregate at an accelerating rate as the animal aged, illustrating an age-dependent collapse of proteostasis in a vulnerable system. Both Hsf-1 and daf-16 slowed this aging phenomenon in genetic experiments. This suggests that aggregation-prone proteins lose function during aging because protein homeostasis fails. This research also suggests therapeutic interventions targeted to small-molecule enhancers of key proteostasis factors, such as Hsf-1, or more specific members of chaperone networks. A commonly proposed mechanism holds that age-related changes in mitochondria lower ATP levels sufficiently to starve proteostatic systems such as chaperones and degradative enzymes. However, this has not been shown in vivo in mammalian models. Moreover, mammalian neurons have significant reserve capacities and alternative sources for ATP generation. They can maintain fairly stable ATP levels even under prolonged fasting conditions. The interaction of Aβ42 with proteostasis and aging remains to be investigated.

Global changes in proteostasis notwithstanding, any given neurodegenerative disease is marked by a selective vulnerability of specific sets of neurons. Studying which normal functions of the disease protein a given cell type loses can give insight into the pathogenic process of the disease at hand. Recent progress on the polyglutamine disease spinocerebellar ataxia 1 (SCA1) is a case in point. This progressive, fatal, autosomal-dominant disease is caused by GAC repeats in the gene encoding ataxin-1, and Purkinje cells in the cerebellar cortex are primarily affected. While the disease features characteristic nuclear inclusions that are positive for ubiquitin, proteasome subunits, and chaperones, research is increasingly shifting from studying toxicity of misfolded aggregates to a newer focus on lost normal functions. Ataxin-1 is widely expressed throughout the brain, yet Purkinje cells degenerate selectively early on even though they are not even among the highest expressors of the gene. Series of mouse models have led to consensus that the disease is a consequence of the expanded protein, not the RNA, and that the large microscopic inclusions of misfolded ATXN1 play less of a role in causing the disease than does the normal function of ATXN1.

ATXN1 is a nuclear protein that interacts with transcription factors. Developmental studies yielded clues to their importance. They showed that transgenic mice developed much more severe disease phenotypes if the ATXN1 polyQ transgene began to be expressed during a specific window of 3 postnatal weeks, when Purkinje cells grow and mature their dendritic trees. If the Purkinje cells were allowed to develop normally during these 3 weeks without expressing the transgene yet, the mice later on were protected from its effects. Subsequent microarray studies examining gene expression during this time window pointed to a selective loss of the RORα gene in Purkinje cells in mutant ATXN1-182Q mice (Serra et al., 2006). RORα forms part of a transcriptional regulation complex that also contains the protein TIP60. This protein interacts with ataxin-1 to destabilize the RORα complex. Further study led to the hypothesis that ataxin-1 normally is part of a transcriptional complex at the promoter sites of various RORα-mediated genes, and that the interaction of mutant ataxin-1 with TIP60 somehow leads to the eventual degradation of this complex so that the respective target genes cannot be expressed. RORα is more highly and specifically expressed in Purkinje cells than is ataxin-1, and the loss of its function might explain part of the characteristic neuronal vulnerability in this disease. The idea is that loss of RORα-mediated gene expression during a time of intense postnatal development stunts the growth of this class of neurons and makes them vulnerable to insults later in life.

This could be explored as a possible general principle in other neurodegenerative diseases, as well. The hypothesis holds that, more generally, alterations in folding due to expanded polyQ tracts or other aggregation-prone molecular characteristics can affect the ability of the protein to function normally, and these functional changes occur long before microscopic pathologies and symptoms show up. These changes can be developmental, leaving neurons prone to age-related insults later.—Gabrielle Strobel.

See also Part 1, Part 2, Part 4, and Part 5.

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References

News Citations

  1. Enabling Technologies for Alzheimer Disease Research: Seventh Bar Harbor Workshop, 2007, Part 1
  2. Enabling Technologies for Alzheimer Disease Research: Seventh Bar Harbor Workshop, 2007, Part 2
  3. Enabling Technologies for Alzheimer Disease Research: Seventh Bar Harbor Workshop, 2007, Part 4
  4. Enabling Technologies for Alzheimer Disease Research: Seventh Bar Harbor Workshop, 2007, Part 5
  5. SfN: Return of the Other—Tau Is Back, Part 3
  6. Shaping Up Amyloid Toxicity: Does It Compute?

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Other Citations

  1. protein misfolding

External Citations

  1. bortezomib

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