Huntingtin

Gene

The 5'-end (five prime end) of the HTT gene has a sequence of three DNA bases, cytosine-adenine-guanine (CAG), coding for the amino acid glutamine, that is repeated multiple times. This region is called a trinucleotide repeat. The usual CAG repeat count is between seven and 35 repeats.

The HTT gene is located on the short arm (p) of chromosome 4 at position 16.3, from base pair 3,074,510 to base pair 3,243,960.

Structure

The Huntingtin (HTT) protein is a large, predominantly α-helical molecule composed of 3,144 amino acids and weighing approximately 348kDa in its canonical form. Its structure is organized into three major domains: the amino-terminal domain, the carboxy-terminal domain, and a smaller bridge domain that connects the two. Both the amino- and carboxy-terminal regions are characterized by multiple HEAT repeats (named for Huntingtin, Elongation factor 3, Protein phosphatase 2A, and lipid kinase TOR), which are arranged in a solenoid or superhelical fashion and play a crucial role in mediating protein-protein interactions. The bridge domain contains various types of tandem repeats and helps maintain the structural connection between the larger domains. The highly variable N-terminal segment of huntingtin contains the polyglutamine (polyQ) tract—expanded in Huntington's disease—which is often intrinsically disordered and not fully resolved in high-resolution structures. Huntingtin's flexible, extended architecture is stabilized when complexed with HAP40, a partner protein, allowing the protein to function as a scaffold and interaction hub in the cell.

In recent years, multiple research groups have managed to resolve the 3D structure of full-size HTT using cryogenic electron microscopy cryoEM. This revealed the 3D architecture of the various helical HEAT repeat domains that make up the protein's native fold, as illustrated in the figure to right. However, up to 25% of the protein chain was not visible in the structure, due to flexibility. This notably included the N-terminal region affected by mutations in Huntington's disease, as discussed below.

Function

The function of huntingtin (Htt) is not well understood but it is involved in axonal transport. Huntingtin is essential for development, and its absence is lethal in mice. The protein has no sequence homology with other proteins and is highly expressed in neurons and testes in humans and rodents. Huntingtin upregulates the expression of brain-derived neurotrophic factor (BDNF) at the transcription level, but the mechanism by which huntingtin regulates gene expression has not been determined. From immunohistochemistry, electron microscopy, and subcellular fractionation studies of the molecule, it has been found that huntingtin is primarily associated with vesicles and microtubules. These appear to indicate a functional role in cytoskeletal anchoring or transport of mitochondria. The Htt protein is involved in vesicle trafficking as it interacts with HIP1, a clathrin-binding protein, to mediate endocytosis, the trafficking of materials into a cell. Huntingtin has also been shown to have a role in the establishment in epithelial polarity through its interaction with RAB11A.

Interactions

Huntingtin has been found to interact directly with at least 19 other proteins, of which six are used for transcription, four for transport, three for cell signalling, and six others of unknown function (HIP5, HIP11, HIP13, HIP15, HIP16, and CGI-125). Over 100 interacting proteins have been found, such as huntingtin-associated protein 1 (HAP1) and huntingtin interacting protein 1 (HIP1), these were typically found using two-hybrid screening and confirmed using immunoprecipitation.

Huntingtin has also been shown to interact with:

• HIP2,
• MAP3K10,
• OPTN,
• PRPF40A,
• SETD2,
• TRIP10,
• ZDHHC17.

Clinical significance

Huntington's disease

Main article: Huntington's disease

Huntington's disease (HD) is caused by a mutated form of the huntingtin gene, where excessive (more than 36) CAG repeats result in formation of an unstable protein. These expanded repeats lead to production of a huntingtin protein that contains an abnormally long polyglutamine tract at the N-terminus. This makes it part of a class of neurodegenerative disorders known as trinucleotide repeat disorders or polyglutamine disorders. The key sequence which is found in Huntington's disease is a trinucleotide repeat expansion of glutamine residues beginning at the 18th amino acid. In unaffected individuals, this contains between 9 and 35 glutamine residues with no adverse effects. However, 36 or more residues produce an erroneous mutant form of Htt, (mHtt). Reduced penetrance is found in counts 36–39.

N-terminal fragments of mHtt have been discovered in Huntington's disease patients. These fragments can be generated by protease enzymes that cut this elongated protein into fragments. Moreover, recent research has identified aberrant splicing to affect the mutant gene products, yielding fragments that coincide with the first exon of the protein. These protein fragments are observed to form abnormal clumps, known as neuronal intranuclear inclusions (NIIs), inside nerve cells, and may attract other, normal proteins into the clumps. The characteristic presence of these clumps in patients was thought to contribute to the development of Huntington disease. However, later research raised questions about the role of the inclusions (clumps) by showing the presence of visible NIIs extended the life of neurons and acted to reduce intracellular mutant huntingtin in neighboring neurons. One confounding factor is that different types of aggregates are now recognised to be formed by the mutant protein, including protein deposits that are too small to be recognised as visible deposits in the above-mentioned studies. The likelihood of neuronal death remains difficult to predict. Likely multiple factors are important, including: (1) the length of CAG repeats in the huntingtin gene and (2) the neuron's exposure to diffuse intracellular mutant huntingtin protein. NIIs (protein clumping) can be helpful as a coping mechanism—and not simply a pathogenic mechanism—to stem neuronal death by decreasing the amount of diffuse huntingtin. This process is particularly likely to occur in the striatum (a part of the brain that coordinates movement) primarily, and the frontal cortex (a part of the brain that controls thinking and emotions). Further, it is possible the pathogenic mechanism lay more with the RNA transcripts and their potential CAG repeats to exhibit RNAi than with the actual huntingtin protein itself.

People with 36 to 40 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with more than 40 repeats will develop the disorder during a normal lifetime. When there are more than 60 CAG repeats, the person develops a severe form of HD known as juvenile HD. Therefore, the number of CAG (the sequence coding for the amino acid glutamine) repeats influences the age of onset of the disease. No case of HD has been diagnosed with a count less than 36.

As the altered gene is passed from one generation to the next, the size of the CAG repeat expansion can change; it often increases in size, especially when it is inherited from the father. People with 28 to 35 CAG repeats have not been reported to develop the disorder, but their children are at risk of having the disease if the repeat expansion increases.

In the pathogenesis of the disease, there is further somatic expansion of CAG repeats. It takes decades to reach 80 repeats, then years to reach 150 repeats. Beyond 150, cellular toxicity start to manifest. Over months, the neuron slowly loses its cell identity until cell death pathways are activated.

Mitochondrial dysfunction

Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Mutant huntingtin (mHtt) plays a key role in mitochondrial dysfunction involving the inhibition of mitochondrial electron transport, inhibition of mitochondrial import processes, higher levels of reactive oxygen species and increased oxidative stress. The promotion of oxidative damage to DNA may contribute to Huntington's disease pathology.

References

References

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