Huntingtin Protein and Protein Aggregation
What causes the onset of HD? Current research shows that there is an abundance of information to be learned regarding the genetic origins of HD. Let’s trace HD’s beginnings from the molecular level, exploring the relationships between a gene, a protein, aggregation “clumps,” neural cell death, and the disease itself.
An Explanation of Terms:
Please note that although “Huntington’s disease” is spelled with an “o,” the correct spelling of the protein involved is “huntingtin” with an “i.” The scientific literature on HD refers to the gene as both the “Huntington gene” and the “huntingtin gene.” For the purposes of this website, we will refer to the gene as the “Huntington gene” or the “HD gene.”
Recall that a gene is a section of DNA made up of four different nucleotide bases, abbreviated by the letters A, T, C, and G. The order of these bases determines the protein “product” of the gene. (To read more about DNA, click here.) Everyone has a gene that codes for huntingtin protein, a protein found in the cells of the body, which we will discuss later. Towards the beginning of this gene, the three-letter codon sequence C-A-G is repeated a few times. Each C-A-G sequence codes for the amino acid glutamine, a protein building block. People with HD simply have an increased number of these C-A-G repeats toward the beginning of their Huntington gene, coding for an excess number of glutamines in their huntingtin protein. This glutamine extension in altered huntingtin leads to further problems, as we will see.
What is the function of huntingtin protein?^
Although researchers are still investigating huntingtin protein’s exact purpose, it appears to play a critical role in nerve cell function. Huntingtin regularly interacts with proteins found only in the brain. Thus, altered huntingtin is most disruptive to nerve cells, even though it is found throughout the body. For more information, click here.
How can the structure of huntingtin be altered?^
The altered form of huntingtin protein has been found in the autopsied brains of patients who have died of HD. Normal, functioning huntingtin protein contains 10-35 glutamines. (Click here to read more about glutamine expansion numbers.) In contrast, altered HD huntingtin protein (called “Htt” by researchers), contains 40 or more glutamine repeats, resulting from the genetic mutation discussed above. The extended number of glutamine repeats in Htt characterizes HD as one of nine polyglutamine expansion disorders. (To read more about these disorders, click here.)
Because glutamine is a polar, or “charged” molecule, the overabundance of glutamine causes links to form within and between proteins. Htt molecules “stick” to one another, forming strands that are held together by hydrogen bonds. Rather than folding into functional proteins, they develop into tangled, rigid groupings known as protein aggregates. (See Figure P-1.) These fibrous protein aggregates accumulate and interfere with nerve cell function by entrapping key cell regulatory factors. Researchers are exploring whether protein aggregates are a cause or a consequence of neurodegenerative diseases such as HD. Are aggregates always harmful, causing nerve cell dysfunction and death, or are they simply part of a cellular defense mechanism? We will explore this question throughout this section.
In a process similar to the formation of aggregates, the excess glutamines in Htt can lead to a type of protein bundling known as neuronal inclusions (NI), or inclusion bodies. NIs initially form at the axons and dendrites of nerve cells in specific areas of the human brain, producing the damaged neurons characteristic of HD. Subsequently, an enzyme cuts Htt into smaller fragments which enter the nerve cell nuclei, forming more clumps at the centrosomes. (See Figure P-2.)
Neuronal inclusions cause problems for the cell. They can cause significant changes in cell structure, “trap” and interfere with the normal production of other proteins, and ultimately become toxic to the nerve cell. Thus, the formation of NIs and the neurodegenerative symptoms of HD are clearly linked.
Does mutant huntingtin protein itself kill nerve cells?^
Not directly. As we have seen, one way Htt indirectly leads to nerve cell damage and toxicity is through the formation of protein aggregates and neuronal inclusions. These structures can interfere with several crucial cellular proteins and systems. Researchers have explored a number of hypotheses regarding the mechanisms by which huntingtin aggregates cause cell dysfunction in HD.
Altered Huntingtin and CREB-binding Protein^
Recent studies have shown that altered huntingtin can “kidnap” smaller proteins from their usual locations, preventing them from functioning normally within nerve cells. A recent Johns Hopkins University study showed that Htt entangles and inhibits CREB-binding protein (also known as CBP), a smaller regulatory protein that is key for cell survival. CBP has its own tract of 18 glutamines, and these glutamines interact directly with the expanded Htt glutamine chain. Huntingtin aggregates pull CBP away from its normal position alongside the DNA in the nucleus. Live mouse models with altered huntingtin and autopsied brains of patients who have died of HD show very reduced amounts of CBP, suggesting that it has been pulled away from the DNA and sequestered by the altered huntingtin.
Once seized, CBP is out of service. It can no longer accomplish its normal function of activatingtranscription or “turning on” genes for survival pathways. Fewer proteins are produced, ultimately leading to nerve cell death. However, researchers were able to fully halt and reverse this degenerative process in the laboratory. This reversal was accomplished by inserting an engineered form of CBP that did not have glutamine repeats. Since Htt interacts directly with the CBP’s glutamines, the modified CBP was not recognized by Htt. The modified CBP was not sequestered in huntingtin inclusion bodies, and the nerve cells survived.
In addition, other compounds known as histone-deacetylase inhibitors (HDAC inhibitors) have been shown to compensate for the negative effects of CBP. It has been shown in fruit flies that HDAC inhibitors reduce the lethal effects of the altered huntingtin. These developments indicate potential targets for new drug treatments, but further research is still needed.
Altered Huntingtin and the Ubiquitin-Proteasome System^
Normal cells have a mechanism for quality control called the ubiquitin-proteasome system (UPS). The UPS is a protein-chaperone system which tags misfolded or damaged proteins for re-folding, or more commonly, for degradation. In contrast, protein aggregates resulting from the altered HD gene have a high molecular mass and are sequestered to the aggresome region of a cell. (See Figure P-3 above.)
Why is the altered form of huntingtin not broken down by the cell, as normally occurs with irregular proteins?^
A recent Stanford University study suggests that protein aggregates impair UPS function, another explanation for how these huntingtin bundles can lead to the death of neurons. Large clumps of defective protein accumulate and scar neurons, which then cannot survive and reproduce. This conclusion about the relationship between protein aggregation, UPS function, and neurodegenerative disease was drawn from an experiment involving mutant cystic fibrosis protein and mutant huntingtin protein. Both of these proteins contain a polyglutamine repeat sequence, giving them the tendency to aggregate. Within human embryonic kidney cells, proteins were tagged with a special form of green fluorescent protein (GFP) which glows only when proteins remain intact. Throughout the experiment, mutant, aggregation-prone proteins continued to glow, showing that they were resistant to degradation, and thus, that the UPS system was stalled. Fluorescence was higher in cells containing aggregates than in cells without, suggesting that protein aggregation led to the UPS disruption.
What is the process by which protein aggregates interfere with the UPS system?^
Well, one clue is that levels of the normal protein ubiquitin are not diminished, and it continues to tag mutant huntingtin for degradation. Thus, it has been suggested that the abnormal proteins clog the other component of the UPS system, the proteasomes. Proteasomes are “master controllers” which normally break down proteins that have been tagged by ubiquitin. Protein aggregates may occupy the proteasomes, inhibiting their normal activity.
Future research in this area may focus on how and why the proteasomes shut down in the presence of protein aggregates. Answers to these questions may lead to treatments that can dissolve aggregate proteins or prevent them from forming, if it is proven that they are indeed only harmful.
Are huntingtin aggregates a cause or consequence of HD?^
This remains a matter of some debate, as the precise role of aggregates in HD is still unclear. On one hand, large inclusions of aggregates seem to be correlated with the development of HD, which suggests a toxic role. There is evidence that huntingtin aggregates accumulate in the nuclei of cells, where they interfere with the process of transcription, and that they also affect other cellular processes such as axonal transport in neurons. A study by Chang et al showed that aggregates can block the movement of mitochondria in neurons, consequently impairing energy production and metabolism. In addition, the previously mentioned Stanford study concludes that aggregates themselves can contribute to cell toxicity by impairing the UPS system. In a vicious cycle, the aggregates inhibit the very pathway that destroys them. Hence, they continue to build up, resulting in further impairment of UPS function. This accumulation of mutant proteins is gradual, which may explain the late onset of HD.
However, there is also much evidence to support the idea that aggregates do not cause HD, but that they are rather an attempt by cells to isolate toxic cell fragments that result from HD, in order to prevent these fragments from causing too much harm. A 2005 study conducted by Slow et al. demonstrated that HD mice with frequent and widespread aggregates did not display any evidence of neuronal dysfunction or degeneration, indicating that aggregation may not be sufficient to cause neurodegeneration. Furthermore, another study conducted by Arrasate et al. demonstrated that aggregates are not directly correlated with neuronal death, and in fact neurons containing aggregates have been shown to survive significantly longer than neurons without aggregates. This suggests that aggregates may actually play a neuroprotective role in HD, acting as a cellular coping mechanism to temporarily store toxic fragments before they are degraded by the proteasome.
One way to reconcile these opposing theories is that aggregate formation does in fact decrease toxicity, but that the UPS system also needs to degrade these aggregates properly and clear toxic fragments from the cell. In other words, aggregate formation might be working together with the UPS system to reduce the levels of toxic mutant huntingtin. Additional research on how to enhance the ability of the UPS system to degrade aggregates may be important for identifying therapeutic targets.
Future Challenges for Huntingtin Research^
Clearly, we have much to learn from exploring the relationships between the Huntington gene, huntingtin protein, protein aggregation, and neural cell death that characterize HD. The effects of HD huntingtin protein on the CBP and UPS pathways are examples of molecular-level problems leading to neural cell death. Future research in this area will likely continue to explore the following questions:
- What is the process by which the proteins aggregate?
- How can the cell get rid of these aggregates?
- What other protein-protein interactions are involved in this disease?
Answers to these questions may point to further possibilities for treatment of HD.