Mechanisms of Protein-Folding Diseases

Protein Misfolding and Disease: A Comprehensive Overview

The Fundamental Problem of Protein Folding

Proteins as Molecular Machines: Proteins are essential molecular machines controlling vital cellular functions.
Crucial for Function: For a protein to function correctly, it must achieve its proper three-dimensional (3D) conformation and locate itself in the correct cellular compartment.
Complexity and Susceptibility to Error: Although protein folding has intrinsic biophysical properties, the process is highly complex and prone to errors. This intricacy, involving multiple chaperone and degradation systems, provides numerous opportunities for misfolding.
Thermodynamic Stability Challenge: The correct folding of a protein often yields only a modest free-energy gain (generally between $-3$ and $-7$ kcal/mol) compared to its countless potential misfolded states. This can sometimes favor misfolded configurations.
Impact of Mutations: Many misfolded proteins involved in disease contain one or more mutations that destabilize the correct fold or stabilize a misfolded state.
The Crowded Cellular Environment: In vivo, protein folding is exacerbated by the cell's crowded environment, where proteins undergo constant high-energy collisions with neighboring molecules, making it difficult to achieve or maintain correct conformations (Ellis and Minton, 2006).
Consequences of Misfolding: Both failure to achieve correct conformations and stable adoption of incorrect ones can lead to disease.

Cellular Compartments and Associated Folding Challenges

Eukaryotic Cell Compartmentalization: Protein folding occurs in various distinct compartments in eukaryotic cells, including:
- Small, specialized organelles (e.g., mitochondria, peroxisomes).
- Massive compartments (e.g., endoplasmic reticulum (ER) for membrane and secreted proteins).
- Cytosol.
- Nucleus.
Diverse Chemical Natures: The varied chemical environments of these compartments present different protein-folding challenges that cells must prevent and address.

Cellular Mechanisms for Combating Protein Folding Problems (Protein Homeostasis)

Cells employ multiple strategies to manage protein folding challenges:

1. Chaperone Systems

Role: Assist proteins in achieving their correct fold or help misfolded proteins regain their proper conformation.
Expression: Chaperones are constitutively expressed and further induced in response to the accumulation of unfolded proteins.
Compartment-Specific Responses:
- Endoplasmic Reticulum (ER): The unfolded protein response (UPR) is activated.
- Nuclear and Cytosolic Compartments: The heat-shock response (HSR) is activated.
- (Other organelles have less characterized responses.)
Beyond Emergency Response: These responses, initially seen as emergency mechanisms for sudden stresses, are now known to constantly address minor perturbations in protein homeostasis and play vital roles in initial folding and refolding (Hartl et al., 2011).

2. Degradation Pathways

Role: Remove misfolded proteins that cannot be properly refolded.
Key Systems: Examples include the proteasome, autophagy, and ER-associated degradation (ERAD) (Nedelsky et al., 2008; Smith et al., 2011; Varshavsky, 2012).
Disease Link: Unsurprisingly, dysfunction in any of these pathways can lead to protein-misfolding diseases.

Historical Context: Sickle Cell Anemia

First Known Molecular Mechanism: Sickle cell anemia was the first inherited human disease with a known molecular mechanism and the first recognized protein-misfolding disease.
Genetic Basis: A single point mutation changes glutamic acid to valine in the eta-globulin chain of hemoglobin (Ingram, 1957; Hunt and Ingram, 1959).
Conformational Change: In the deoxygenated environment of tissue capillary beds (Gibson and Ellory, 2002), the protein changes conformation, exposing a hydrophobic patch.
Pathology: This leads to polymerization in individuals homozygous for the mutation, reducing red blood cell elasticity, causing severe pain, extensive tissue destruction, and anemia.
Protective Allele: The heterozygous state provides protection against malaria, leading to high allele prevalence in some African populations (Aidoo et al., 2002).
Complexity: The relationship between genetic changes, protein misfolding, and disease is often more complex than in sickle cell anemia.

Protein Misfolding and Human Disease: Five Mechanisms

Protein misfolding is implicated in hundreds of diseases, forming the basis for most non-infectious diseases. This section illustrates five diverse mechanisms and their therapeutic implications.

1. Improper Degradation

Mechanism: Cellular degradation systems (e.g., ERAD, autophagy), while essential for clearing non-functional proteins, can be overactive, leading to the destruction of mutant proteins that still retain some beneficial function.
Cystic Fibrosis (CF):
- Cause: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane chloride channel.
- Most Common Mutation: Deletion of phenylalanine at position $508$ ( $riangle F508$ ) in CFTR.
- Effect: This mutation causes the protein to misfold and be targeted for degradation, even though a fraction might be partially functional if allowed to mature (Qu et al., 1997).
- Therapeutic Approaches:
  - Inhibiting Chaperones: Disrupting chaperone systems can allow mutant CFTR to escape degradation. For example, knockdown of AHA1 (a co-chaperone that works with HSP90) stabilizes $riangle F508$ CFTR and restores partial function (Wang et al., 2006).
  - Blocking Degradation Pathways: Blocking CHIP (an Hsc70 co-chaperone that aids in ubiquitylation and degradation of mutant CFTR) could also allow more CFTR to mature and function (Meacham et al., 2001).
Gaucher's Disease:
- Classification: The most common lysosomal storage disease (Futerman and van Meer, 2004; Cox and Cachón-González, 2012).
- Cause: Mutations in eta-glucosidase (also known as eta-glucocerebrosidase), a lysosomal enzyme involved in glucosylceramide metabolism.
- Effect: Defects lead to intracellular accumulation of the enzyme's substrate, particularly in white blood cells. Symptoms vary widely (e.g., bone lesions, enlarged spleen/liver) (Grabowski, 2008), potentially linked to the extent of ER degradation of eta-glucosidase (Ron and Horowitz, 2005).
- Therapeutic Approaches:
  - Upregulating Chaperones: Hypothesis: Upregulation of chaperones assisting eta-glucosidase folding could be beneficial (Sawkar et al., 2006).
  - Pharmacological Chaperones: Small-molecule drugs that mimic protein chaperones by directly binding and stabilizing the enzyme, allowing it to reach the lysosome. At the lysosome, the physiological substrate (glucosylceramide) displaces the chaperone, activating the enzyme (Sawkar et al., 2002; Sawkar et al., 2005).
  - Current Treatment: Enzyme replacement therapy is a primary option but is expensive and requires intravenous delivery.
  - Future Hope: Small-molecule-based therapy (potentially cheaper and easier to administer) offers an alternative strategy.

2. Improper Localization

Mechanism: Mutations that destabilize a protein's correct fold can lead to improper subcellular localization, resulting in two types of toxicity:
- Loss of Function: The protein fails to perform its role at its correct location.
- Gain of Function Toxicity: The protein accumulates in an incorrect location, causing damage.
oldsymbol{oldsymbol{ ext{}}}oldsymbol{ ext{}} ext{1-Antitrypsin} oldsymbol{ ext{}} oldsymbol{ ext{}}( ext{AAT}) ext{ Deficiency: A dual toxicity example (Perlmutter, 2011).
- Recessive Loss-of-Function: Leads to emphysema due to lack of secreted AAT.
- Dominant Gain-of-Function: Causes liver damage due to accumulation of misfolded AAT.
- Mechanism: Mutant AAT fails to fold correctly and is retained in the ER of hepatocytes (its synthesis site). Unlike other misfolded proteins, it is not degraded and accumulates, causing liver damage (Lomas et al., 1992; Hidvegi et al., 2005).
- Lung Pathology: Non-secreted AAT cannot inhibit proteases (e.g., neutrophil elastase) in the lung, leading to extensive damage to lung connective tissue.
- Therapeutic Approaches:
  - Lung Damage: Can be managed with enzyme replacement therapy (Mohanka et al., 2012).
  - Liver Accumulation: A greater challenge. Drugs that enhance macroautophagy (e.g., rapamycin, carbamazepine) alleviate hepatic toxicity by promoting degradation of aggregates (Hidvegi et al., 2010).
  - Direct Blockage: Directly blocking the aggregation of mutant AAT (Skinner et al., 1998; Mallya et al., 2007).

3. Dominant-Negative Mutations

Mechanism: A mutant protein antagonizes the function of the wild-type (WT) protein, causing a loss of protein activity even in heterozygotes.
Epidermolysis Bullosa Simplex:
- Classification: An inherited connective tissue disorder.
- Cause: Mutant forms of keratin proteins KRT5 and KRT14 lead to severe skin blistering upon injury (Chamcheu et al., 2011a).
- Mechanism: Keratin forms intermediate filaments providing epidermal structure. Disease-associated mutations cause keratin to misfold and aggregate, particularly under mechanical stress (Russell et al., 2004; Werner et al., 2004). In heterozygotes, filaments contain both WT and mutant proteins, and the mutant proteins compromise the function of the entire filament.
- Therapeutic Approaches:
  - Chemical Chaperones: Identified to prevent aggregation of mutant keratin and alleviate symptoms (Chamcheu et al., 2012).
  - 4-Phenylbutyrate (4-PBA): A chemical chaperone that causes degradation of aggregated keratin, possibly by increasing cellular concentrations of protein chaperones (Chamcheu et al., 2011b). Treatment with 4-PBA decreases aggregated keratin and increases HSP70 colocalization with remaining keratin, suggesting HSP70 activation for degradation. Notably, 4-PBA is already approved for other disorders (Maestri et al., 1996), facilitating its development for this disease.
p53 in Cancer:
- p53 Function: A homotetrameric transcription factor crucial for regulating pathways maintaining genome integrity (apoptosis, DNA damage repair, cell cycle regulation, metabolism) (Freed-Pastor and Prives, 2012).
- Mutations in Cancer: Mutations in p53 are among the most common genetic alterations in cancer (Friedman et al., 1993).
- Normal Regulation: In the absence of genotoxic stress, p53 is rapidly degraded by the proteasome in an MDM2 ubiquitin ligase-dependent manner (Kubbutat et al., 1997). Stress stabilizes p53, allowing it to activate target genes.
- Oncogenic Mutations: Many cancer-associated mutations disrupt the p53 core domain, preventing its correct folding, with two main consequences:
  - Dominant-Negative Effect: Mutant p53 can still associate with WT p53 monomers, but the resulting tetramer is dysfunctional, even if WT copies are present (Milner and Medcalf, 1991; Milner et al., 1991). In heterozygotes, most tetramers become dysfunctional.
  - Stabilization: Mutant p53 cannot interact with MDM2, preventing degradation. It is also stabilized by chaperones like HSP90, leading to inappropriate accumulation and further reducing the chance of forming functional WT-only tetramers.
- Therapeutic Approaches:
  - Nutlins: Small molecules in clinical trials for p53-dysfunction-dependent cancers. They prevent MDM2 from interacting with and degrading WT p53, increasing the probability of forming functional WT tetramers (Vassilev et al., 2004).
  - Mutant p53 Reactivators: Small molecules that directly bind mutant p53 and restore its function. For example, pk7088 binds and stabilizes the p53 mutant Y220C, restoring its transcriptional functions to WT levels (Liu et al., 2013). This highlights the potential for personalized medicine in cancer treatment, as specific compounds can target specific mutations.

4. Gain of Toxic Function

Mechanism: Protein conformational changes lead to a protein acquiring a new, toxic function.
Apolipoprotein E (APOE4) in Alzheimer's Disease (AD):
- Association: At least one copy of the APOE4 allele is found in 65-80 ext{%} $of individuals with AD (Farrer et al., 1997).</li> <li><strong>Mechanism:</strong> The APOE4 polymorphism stabilizes an altered conformational fold, involving an extra salt bridge (between Arginine$ 61 $and Glutamic acid$ 255 $) that compromises its extended domain structure (Dong et al., 1994; Dong and Weisgraber, 1996).</li> <li><strong>Toxic Effects:</strong> This interaction changes APOE4's lipid affinity (Dong et al., 1994; Dong and Weisgraber, 1996), disrupts mitochondrial function (Chen et al., 2011), impairs neurite outgrowth (Nathan et al., 1994), and is associated with increased levels of A$ eta$$, the peptide that aggregates in AD brains (Ma et al., 1994).
- Therapeutic Approaches:
  - Structure Correctors: Small molecules that prevent the formation of the extra salt bridge. A FRET-based assay identified structure correctors that rescued APOE4 misfolding and its detrimental effects on mitochondrial dysfunction and neurite outgrowth (Brodbeck et al., 2011).
Oncogenic Proteins in Cancer (e.g., v-SRC, BCR-ABL, BRAF):
- Mechanism: Many oncogenic proteins acquire novel pathological functions through mutation (e.g., mutant v-SRC lacks its normal self-inhibitory phosphorylation site and promotes uncontrolled cell proliferation).
- Chaperone Dependence: While constitutively active, these oncogenic mutants (e.g., v-SRC) are often less stable than their WT counterparts (c-SRC). They exploit the HSP90 chaperone system, which acts as a folding