5.3 Misfolding, aggregation, and associated diseases

Protein misfolding and aggregation are critical issues in cellular health. When proteins fail to fold correctly, they can clump together, forming toxic aggregates that disrupt normal cell function. This process is linked to numerous neurodegenerative diseases, including Alzheimer's and Parkinson's.

Understanding protein misfolding is crucial in the broader context of protein structure and folding. It highlights the importance of proper folding for protein function and the consequences when this process goes awry. Exploring misfolding and aggregation provides insights into potential therapeutic strategies for related diseases.

Protein misfolding and aggregation

Causes and consequences of protein misfolding

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• Protein misfolding occurs when a protein fails to achieve or maintain its native, functional conformation caused by genetic mutations, environmental factors, or errors in the protein folding process
• Misfolded proteins often expose hydrophobic regions that are normally buried in the protein's interior leading to aggregation and the formation of insoluble protein deposits
• Protein aggregation can cause cellular dysfunction by disrupting normal protein function, inducing oxidative stress, and overwhelming the cellular protein quality control mechanisms
• Misfolded and aggregated proteins can exhibit gain-of-function toxicity, where they acquire new, harmful functions, such as the formation of pore-like structures that disrupt cellular membranes
  • These pore-like structures can lead to uncontrolled ion flux and disrupt cellular homeostasis
  • Gain-of-function toxicity can also involve the sequestration of essential cellular proteins into aggregates, impairing their normal functions
• The accumulation of misfolded and aggregated proteins is a hallmark of many neurodegenerative diseases (Alzheimer's, Parkinson's, and Huntington's diseases)
  • These diseases are characterized by the progressive loss of specific neuronal populations and the presence of protein aggregates in affected brain regions
  • The specific proteins involved in aggregation vary among different neurodegenerative diseases (amyloid-beta in Alzheimer's, alpha-synuclein in Parkinson's, huntingtin in Huntington's)

Chaperones in protein folding

Molecular chaperones and their functions

• Molecular chaperones (heat shock proteins or HSPs) assist in the proper folding of newly synthesized proteins and help to refold misfolded proteins
  • HSPs are classified by their molecular weight (HSP60, HSP70, HSP90) and have diverse functions in protein folding and quality control
  • Examples of HSPs include HSP70, which binds to nascent polypeptide chains and assists in their folding, and HSP90, which stabilizes and activates a wide range of client proteins
• Chaperones recognize and bind to exposed hydrophobic regions of misfolded proteins preventing their aggregation and promoting their refolding or degradation
  • The hydrophobic regions of misfolded proteins are typically buried in the native state but become exposed during misfolding
  • Chaperones use ATP-driven conformational changes to bind and release misfolded proteins, facilitating their refolding or targeting them for degradation

Cellular stress responses and protein quality control

• The unfolded protein response (UPR) is activated in response to an accumulation of misfolded proteins in the endoplasmic reticulum (ER) leading to increased expression of chaperones and other stress response proteins
  • The UPR involves three main signaling pathways (IRE1, PERK, and ATF6) that work together to reduce protein folding load, increase ER folding capacity, and promote the degradation of misfolded proteins
  • Chronic activation of the UPR can lead to apoptosis if the protein folding stress cannot be resolved, a process that may contribute to neurodegeneration
• The ubiquitin-proteasome system (UPS) selectively degrades misfolded and damaged proteins by tagging them with ubiquitin and targeting them for proteolysis by the proteasome
  • Ubiquitin is a small protein that is covalently attached to target proteins through the action of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes
  • The 26S proteasome is a large, ATP-dependent proteolytic complex that recognizes and degrades ubiquitinated proteins
• Autophagy is a cellular process that degrades misfolded proteins and damaged organelles by sequestering them in double-membrane vesicles called autophagosomes, which then fuse with lysosomes for degradation
  • Autophagy can be divided into three main types: macroautophagy, microautophagy, and chaperone-mediated autophagy
  • Autophagy plays a crucial role in maintaining cellular homeostasis and has been implicated in the pathogenesis of several neurodegenerative diseases

Protein misfolding diseases

Alzheimer's disease

• Alzheimer's disease is characterized by the accumulation of extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein
  • Aβ peptides are generated by the proteolytic cleavage of the amyloid precursor protein (APP) and can misfold and aggregate into oligomers and fibrils leading to neuronal dysfunction and death
    • The cleavage of APP by β-secretase and γ-secretase generates Aβ peptides of varying lengths (Aβ40 and Aβ42)
    • Aβ42 is more prone to aggregation and is the primary component of amyloid plaques
  • Hyperphosphorylation of tau protein causes it to misfold and aggregate into neurofibrillary tangles disrupting the microtubule network and impairing axonal transport
    • Tau is a microtubule-associated protein that stabilizes microtubules and promotes their assembly
    • Hyperphosphorylation of tau leads to its dissociation from microtubules and the formation of insoluble aggregates

Parkinson's disease

• Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intracellular protein aggregates called Lewy bodies, which are primarily composed of misfolded α-synuclein
  • Mutations in the SNCA gene, which encodes α-synuclein, or environmental factors can cause α-synuclein to misfold and aggregate leading to neuronal dysfunction and death
    • α-synuclein is a small, soluble protein that is abundantly expressed in the brain and is involved in synaptic vesicle trafficking
    • Mutations (A53T, A30P, E46K) or multiplications of the SNCA gene can increase the propensity of α-synuclein to misfold and aggregate
  • The spread of misfolded α-synuclein from cell to cell is thought to contribute to the progression of Parkinson's disease pathology throughout the brain
    • Misfolded α-synuclein can be released from neurons and taken up by neighboring cells, where it can seed the misfolding and aggregation of endogenous α-synuclein
    • This prion-like propagation of α-synuclein pathology may explain the stereotypical progression of Parkinson's disease symptoms and pathology

Other protein misfolding diseases

• Huntington's disease is caused by polyglutamine expansions in the huntingtin protein
  • The mutant huntingtin protein contains an expanded tract of glutamine residues (>36 repeats) that causes it to misfold and aggregate
  • The aggregation of mutant huntingtin leads to the formation of intranuclear inclusions and neuronal dysfunction and death, particularly in the striatum and cortex
• Amyotrophic lateral sclerosis (ALS) is associated with the misfolding of SOD1, TDP-43, and FUS proteins
  • Mutations in the SOD1 gene, which encodes superoxide dismutase 1, can cause the protein to misfold and aggregate, leading to motor neuron degeneration
  • TDP-43 and FUS are RNA-binding proteins that can misfold and aggregate in ALS, forming cytoplasmic inclusions and disrupting RNA metabolism
• Prion diseases are caused by the misfolding and aggregation of the prion protein (PrP)
  • The cellular prion protein (PrPC) can misfold into an infectious, pathogenic conformation (PrPSc) that can template the misfolding of other PrPC molecules
  • The accumulation of PrPSc leads to neuronal degeneration and the formation of spongiform changes in the brain, as seen in Creutzfeldt-Jakob disease (CJD) and other prion disorders

Treatment of protein misfolding diseases

Enhancing cellular protein quality control mechanisms

• Enhancing the cellular protein quality control mechanisms (chaperones and the UPS) can help to prevent the accumulation of misfolded and aggregated proteins
  • Small molecules that upregulate chaperone expression or activity (HSP90 inhibitors) are being investigated as potential therapeutics for protein misfolding diseases
    • HSP90 inhibitors (geldanamycin, 17-AAG) can induce the heat shock response and increase the expression of other HSPs, such as HSP70, which can help to refold misfolded proteins
    • Other compounds that activate the heat shock response, such as celastrol and arimoclomol, are also being explored as potential therapies
  • Compounds that enhance proteasome activity or reduce the burden on the UPS (proteasome activators or aggregation inhibitors) may also be effective in treating these disorders
    • Proteasome activators (PA28, USP14 inhibitors) can increase the degradation of misfolded proteins by the UPS
    • Aggregation inhibitors (scyllo-inositol, trehalose) can prevent the formation of protein aggregates and reduce the burden on the UPS

Targeting specific misfolded proteins

• Targeting the specific misfolded proteins involved in each disease (Aβ, tau, or α-synuclein) can help to reduce their aggregation and toxicity
  • Immunotherapies using antibodies that bind to and promote the clearance of misfolded proteins are being developed for Alzheimer's and Parkinson's diseases
    • Aβ immunotherapies (bapineuzumab, solanezumab) aim to reduce the levels of Aβ in the brain by promoting its clearance or preventing its aggregation
    • Tau immunotherapies (ABBV-8E12, gosuranemab) target various epitopes on the tau protein and aim to reduce the formation and spread of neurofibrillary tangles
  • Small molecules that inhibit the aggregation of specific misfolded proteins or disrupt pre-formed aggregates are also being investigated as potential therapeutics
    • Aβ aggregation inhibitors (tramiprosate, curcumin) can prevent the formation of Aβ oligomers and fibrils
    • α-synuclein aggregation inhibitors (anle138b, NPT200-11) can reduce the formation of α-synuclein oligomers and fibrils and prevent their prion-like spread

Reducing oxidative stress and inflammation

• Reducing oxidative stress and inflammation, which can contribute to the pathogenesis of protein misfolding diseases, may help to slow disease progression and protect against neuronal damage
  • Antioxidants and anti-inflammatory compounds (polyphenols, non-steroidal anti-inflammatory drugs or NSAIDs) are being studied for their potential neuroprotective effects in protein misfolding diseases
    • Polyphenols (resveratrol, epigallocatechin gallate) have antioxidant and anti-inflammatory properties and can reduce the production of reactive oxygen species and inflammatory cytokines
    • NSAIDs (ibuprofen, naproxen) can reduce neuroinflammation by inhibiting cyclooxygenase enzymes and reducing the production of pro-inflammatory prostaglandins
  • Strategies to boost endogenous antioxidant defenses, such as the activation of the Nrf2 pathway, are also being explored as potential therapies for protein misfolding diseases
    • Nrf2 activators (dimethyl fumarate, sulforaphane) can increase the expression of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and protect against oxidative stress-induced neuronal damage

Gene therapy approaches

• Gene therapy approaches aimed at reducing the expression of mutant proteins or increasing the expression of protective factors (chaperones or autophagy regulators) are also being explored as potential treatments for protein misfolding diseases
  • RNA interference (RNAi) and antisense oligonucleotides (ASOs) can be used to selectively knockdown the expression of mutant proteins, such as huntingtin in Huntington's disease or SOD1 in ALS
    • RNAi involves the use of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) that bind to and promote the degradation of target mRNAs
    • ASOs are single-stranded DNA molecules that can bind to and promote the degradation of target mRNAs or modulate their splicing
  • Gene delivery of chaperones or autophagy regulators can help to enhance the cellular protein quality control mechanisms and reduce the accumulation of misfolded proteins
    • Viral vectors (adeno-associated virus, lentivirus) can be used to deliver genes encoding HSPs or autophagy-promoting factors (Beclin-1, TFEB) to specific brain regions affected by protein misfolding diseases
    • Non-viral gene delivery methods, such as nanoparticles or exosomes, are also being explored as potential vehicles for the delivery of therapeutic genes or RNAs