Mechanisms of Protein-Folding Diseases

The Protein-Folding Problem and Disease Etiology

• Fundamental Role of Protein Conformation: For proteins to function correctly, they must achieve proper three-dimensional conformation and reside in the correct location within the cell's crowded environment.

• Cellular Quality Control Systems:

  • Chaperone Systems: Multiple systems are required to fold proteins accurately.

  • Degradation Pathways: Systems like the proteasome and autophagy destroy improperly folded proteins that cannot be salvaged.

• Susceptibility to Error: The multi-systemic intricacy of protein folding provides numerous opportunities for errors.

  • Mutations: Genetic mutations frequently lead to misfolded, non-functional protein forms that accumulate within cells.

  • Thermodynamic Stability: Correct folding often involves only a modest free-energy gain (generally $-3 ext{ to } -7 ext{ kcal/mol}$) compared to myriad misfolded states, which can sometimes be thermodynamically favored.

  • Crowded Environment: In vivo, the crowded cellular environment, with constant high-energy collisions, complicates proper protein folding.

• Cellular Compartments for Folding: In eukaryotic cells, protein folding occurs in various distinct compartments, including:

  • Endoplasmic Reticulum (ER) for membrane and secreted proteins.

  • Cytosol and Nucleus.

  • Specialized organelles like mitochondria and peroxisomes.

  • Each compartment's unique chemical nature presents different folding challenges.

• Cellular Responses to Misfolding:

  • Chaperone Induction: Chaperones are constitutively expressed and further induced when unfolded proteins accumulate.

    • In the ER, this is known as the Unfolded Protein Response (UPR).

    • In the nuclear and cytosolic compartments, it's the Heat-Shock Response (HSR).

    • Beyond emergency responses, these are constantly active, aiding initial folding and refolding of misfolded proteins.

  • Degradation Deployment: If refolding fails, systems like the proteasome, autophagy, and ER-associated degradation (ERAD) degrade misfolded proteins.

  • Dysfunction in these pathways can directly lead to protein-misfolding diseases.

• Ubiquity of Protein Misfolding Diseases: Protein misfolding is implicated in the progression of hundreds of diseases, forming the underlying cause for the majority of diseases not initiated by an infectious agent.

• Five Major Mechanisms of Disease Caused by Protein Misfolding: This review illustrates the breadth of the phenomenon through five examples:

  1. Improper degradation.

  2. Mislcalization.

  3. Dominant-negative mutations.

  4. Structural alterations that establish novel toxic functions.

  5. Amyloid accumulation.

Classic Example: Sickle Cell Anemia

• Historical Significance: This was the first known protein-misfolding disease and the first inherited human disease with a known molecular mechanism.

• Genetic Basis: Caused by a single point mutation where glutamic acid in the $\beta$-globulin chain of hemoglobin is replaced by valine.

• Molecular Mechanism:

  • In the deoxygenated environment of tissue capillary beds, the mutant hemoglobin protein undergoes a conformational change.

  • This change exposes a hydrophobic patch.

  • The exposed hydrophobic patches lead to the polymerization of hemoglobin molecules in individuals homozygous for the mutation.

• Pathophysiological Consequences:

  • Reduced elasticity of red blood cells.

  • Causes extreme pain.

  • Leads to extensive tissue destruction and anemia.

• Evolutionary Relevance: The allele for sickle cell anemia is maintained at high levels in several African populations because, in the heterozygous state, it confers some protection against the malaria parasite, which replicates in red blood cells.

Disease Mechanism 1: Improper Degradation

• Mechanism Description: Cellular degradation systems, while essential for removing non-functional misfolded proteins, can sometimes be overactive. This leads to the degradation of mutant proteins that, despite their defects, retain some functionality, exacerbating disease severity.

Cystic Fibrosis (CF)

• Causative Gene: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane chloride channel.

• Most Common Mutation: Deletion of a phenylalanine residue at position $508$ ($\Delta$F508) in CFTR.

• Pathology: The $\Delta$F508 mutation causes the CFTR protein to misfold and be targeted for degradation by the cell's quality control systems.

• Chaperone Involvement: CFTR maturation and degradation require interactions with multiple chaperones and co-chaperones.

• Therapeutic Strategies: Inhibiting specific chaperone systems might allow mutant CFTR to escape degradation and retain partial function.

  • AHA1 Knockdown: Knockdown of AHA1, a co-chaperone that works with HSP90 to influence CFTR maturation, results in $\Delta$F508 CFTR being more stable and partially functional.

  • CHIP Inhibition: CHIP, an HSP70 co-chaperone, aids in the ubiquitylation and subsequent degradation of mutant CFTR; blocking CHIP function could potentially allow more CFTR to mature and function.

Gaucher's Disease

• Disease Type: The most common lysosomal storage disease.

• Causative Gene: A variety of mutations in $\beta$-glucosidase (also known as $\beta$-glucocerebrosidase), a lysosomal enzyme involved in glucosylceramide metabolism.

• Pathology: Defects in this enzyme lead to the intracellular accumulation of its substrate (glucosylceramide), particularly within white blood cells.

• Clinical Variability: Symptoms (e.g., bone lesions, enlarged spleen and liver) show high variability, which is thought to correlate with the extent of $\beta$-glucosidase degradation in the ER.

  • Some fraction of the mutant protein can be correctly processed and trafficked to the lysosome, retaining functionality even in affected individuals.

• Therapeutic Strategies:

  • Chaperone Upregulation: The hypothesis is that upregulating chaperones that assist in the correct folding of $\beta$-glucosidase could be a useful intervention.

  • Pharmacological Chaperones: Small molecules that mimic protein chaperones by directly binding and stabilizing the enzyme's fold. This allows the enzyme to reach its site of activity (the lysosome). At the lysosome, the pharmacological chaperone is displaced by glucosylceramide, allowing the enzyme to become active.

    • These drugs have shown efficacy in cells from Gaucher's patients and some activate the UPR.

  • Comparison to Current Treatments: Enzyme replacement therapy is a main treatment but requires expensive intravenous delivery. Small-molecule-based therapy is a potentially cheaper and easier-to-administer alternative.

Disease Mechanism 2: Improper Localization

• Mechanism Description: Mutations that destabilize a protein's correct fold can prevent its proper trafficking, leading to incorrect subcellular localization.

• Dual Toxicity Consequences:

  • Loss of Function: The protein cannot perform its role at its intended cellular location.

  • Gain-of-Function Toxicity: The mislocalized protein accumulates in an incorrect location, leading to toxic effects.

$\alpha1$-antitrypsin Deficiency

• Protein Role: $\alpha1$-antitrypsin is a secreted protease inhibitor.

• Mutation Effects: When mutated, it causes two distinct disease manifestations:

  • Emphysema: Recessive loss-of-function due to the inability to inhibit proteases (e.g., neutrophil elastase) in the lung, leading to extensive damage to connective tissue.

  • Liver Damage: Dominant gain-of-function due to the accumulation of misfolded protein in the ER of hepatocytes (site of synthesis).

• Pathology Specifics: Mutant forms fail to complete proper folding and are uniquely retained, but not efficiently degraded, in the ER, leading to aggregate accumulation and liver damage.

• Therapeutic Strategies:

  • Lung Damage: Can be managed with enzyme replacement therapy.

  • Liver Accumulation (Greater Challenge):

    • Autophagy Enhancers: Drugs like rapamycin and carbamazepine, which enhance macroautophagy, promote the degradation of aggregates in the liver, alleviating hepatic toxicity.

    • Aggregation Blockers: Other therapies focus on directly blocking the aggregation of mutant $\alpha1$-antitrypsin.

Disease Mechanism 3: Dominant-Negative Mutations

• Mechanism Description: A mutant protein antagonizes the function of the wild-type (WT) protein, resulting in a loss of protein activity even in heterozygous individuals.

Epidermolysis Bullosa Simplex

• Disease Type: An inherited connective tissue disorder.

• Causative Proteins: Mutant forms of keratin proteins KRT5 and KRT14.

• Protein Role: Keratin proteins form long intermediate filaments that provide structural integrity to the epidermis of the skin.

• Pathology: Disease-associated mutations cause keratin proteins to misfold and aggregate, particularly in response to mechanical stress. Since intermediate filaments are constructed from multiple keratin molecules, a heterozygote will produce filaments containing both WT and mutant proteins. The dysfunctional mutant keratin compromises the entire filament, explaining the dominant nature of the disease, leading to severe skin blistering upon injury.

• Therapeutic Strategies:

  • Chemical Chaperones: Recent research has identified chemical chaperones that can prevent the aggregation of mutant keratin and alleviate symptoms.

    • 4-phenylbutyrate (4-PBA): One such compound, 4-PBA, has been found to cause the degradation of aggregated keratin. Its mechanism may involve increasing the cellular concentration of protein chaperones, as it decreases total aggregated keratin and increases HSP70 colocalization with remaining keratin.

    • Drug Development Advantage: 4-PBA is already approved for treating other disorders, streamlining its development pipeline for this application.

p53 Mutations in Cancer

• Protein Role: p53 is a homotetrameric transcription factor responsible for regulating numerous pathways critical for maintaining genome integrity, including:

  • Apoptosis.

  • DNA damage repair.

  • Cell cycle regulation.

  • Metabolism.

• Prevalence: Mutations in p53 are among the most common genetic alterations found in cancer, indicating their far-reaching effects.

• Normal Regulation:

  • In the absence of genotoxic stress, p53 is rapidly degraded by the proteasome, a process dependent on the ubiquitin ligase MDM2.

  • In response to stresses like DNA damage, p53 is stabilized, allowing it to stimulate the transcription of its target genes.

• Oncogenic p53 Mutations (Dominant-Negative Mechanism): Many common oncogenic mutations disrupt the core domain of p53, preventing it from assuming its correctly folded conformation. This leads to a lack of expression of genome-protective genes, increasing cancer risk.

  • Dysfunctional Tetramer Formation: Mutant p53 can still associate with other p53 monomers, but the resulting tetramer does not function correctly, even if a WT copy of p53 is present. This demonstrates the dominant-negative effect, where most tetramers are dysfunctional in a heterozygous state.

  • Stabilization and Accumulation: Mutant p53 is unable to interact with MDM2 and thus escapes rapid degradation. It is further stabilized by binding to chaperones like HSP90. This inappropriate accumulation of mutant p53 makes it less likely that a functional tetramer composed solely of WT p53 will form.

• Therapeutic Strategies:

  • Nutlins: These small molecules are currently in clinical trials for p53-dysfunction-dependent cancers. They prevent MDM2 from interacting with and promoting the degradation of WT p53, thereby increasing the probability of forming functional WT tetramers.

  • Mutant p53 Reactivators: Small molecules have been discovered that directly bind mutant p53 and restore its function.

    • Example: pk7088: This compound binds and stabilizes a specific p53 mutant, Y220C, restoring its transcriptional functions to WT levels. Even though Y220C is only a fraction of p53 mutations, p53 is so frequently mutated that this fraction still represents a large patient population, highlighting the importance of personalized medicine in cancer treatment.

Disease Mechanism 4: Gain of Toxic Function

• Mechanism Description: Protein conformational changes can lead to dominant phenotypes by causing a protein to acquire a new, toxic conformation or activity.

Apolipoprotein E (APOE) in Alzheimer's Disease (AD)

• Protein Role: APOE is a lipid transport molecule.

• APOE4 Allele: At least one copy of the APOE4 allele is found in $\text{65-80\%}$ of individuals with AD.

• Mechanism of Toxicity: The APOE4 polymorphism stabilizes an altered conformational fold in the protein. This is due to an extra salt bridge formation (an interaction between Arginine $61$ and Glutamic acid $255$) that compromises the extended domain structure seen in other APOE alleles.

• Consequences: This aberrant interaction leads to:

  • Changes in APOE4's lipid affinity.

  • Disruption of mitochondrial function.

  • Impairment of neurite outgrowth.

  • Association with increased levels of A$\beta$, the peptide that aggregates in the brains of AD patients.

• Therapeutic Strategies: Targeting the specific conformational change.

  • Structure Correctors: Small molecules that prevent the formation of the extra salt bridge. A FRET-based assay identified such correctors, which not only prevented APOE4 misfolding but also rescued APOE4-associated mitochondrial dysfunction and relieved inhibition of neurite outgrowth.

Oncogenic Kinases in Cancer

• Mechanism Description: Many oncogenic proteins acquire novel pathological functions through mutation, driving various cancers.

• Example: v-SRC: The first identified oncogenic protein, mutant v-SRC (a non-receptor tyrosine kinase), lacks its normal self-inhibitory phosphorylation site, promoting uncontrolled cell proliferation. While constitutively active, v-SRC is less stable than the WT c-SRC protein.

• Role of HSP90: Oncogenic mutants, including v-SRC, exploit the HSP90 chaperone system, which acts as a protein-folding