Arches. Photo by Daniel Chia
HOPES: Huntington's Outreach Project for Education, at Stanford
Feb
03
2014

TRiC and Huntingtin Protein Aggregation

In Huntington’s disease (HD), an abnormal increase in the number of CAG repeats in the mutant Huntington gene corresponds to a long tract of glutamine amino acids in the huntingtin protein (Htt). This excessively long glutamine tract is sticky and leads to the formation of protein aggregates in brain cells. Whether these aggregates are toxic only upon formation or are formed as a cellular defense mechanism against free toxic mutant huntingtin protein (mHtt) is still hotly debated (more information about huntingtin protein aggregation can be found here); however, what is certain is that these protein aggregates are directly linked to neuron dysfunction and death. Moreover, a larger number of CAG repeats is clearly correlated with earlier disease onset and increased severity.

Although these findings suggest that HD is solely a result of the CAG expansion and subsequent protein aggregation, several observations indicate that other factors are also at play. For example, patients with the same number of CAG repeats may exhibit different disease patterns. In addition, there are genes other than the Huntington gene that also contain a CAG repeat region. Some of these genes may have a long region of upwards of 50 CAG repeats, but still produce functional non-harmful proteins (recall that HD can occur in patients with as few as 36 CAG repeats in the Huntington gene).  Evidently, there are other cellular mechanisms, though not well understood, that govern how the expanded number of CAG repeats translates into the toxic behavior of mHtt and aggregates seen in the specific case of HD.

Recently, researchers have found increasing evidence that the cellular mechanisms contributing to neurodegenerative diseases resulting from protein aggregates involve disruptions to the quality control machinery in the cell responsible for identifying and eliminating these toxic proteins. In particular, a protein known as TRiC (TCP-1 ring complex) has been found to be involved in controlling whether proteins with a long glutamine tract like Htt will fold and aggregate.  This article will first review how certain classes of proteins can control protein folding, then go over the case of TRiC and its role in Htt folding, and finally discuss how this knowledge might be helpful for researching HD therapeutics.

Protein Folding and Molecular Chaperones^

To understand how TRiC is involved in huntingtin (Htt) protein aggregation, it is useful to first review the players that make proteins in the cell. First, mRNA instructions are made based on the DNA blueprint in the nucleus, or the control center, of the cell. These instructions are then sent out into the cytoplasm of the cell and are read by structures known as ribosomes, the actual molecular machines that make proteins. Proteins are synthesized as long chains of amino acid building blocks, which are assembled one by one as the ribosome reads the mRNA instructions. For a more detailed breakdown of the protein synthesis process, go here.

However, proteins are not functional as a linear chain; they need to take on a certain shape or conformation in order to exert their proper functions. To assist in this process known as protein folding, proteins known as molecular chaperones bind to chains of amino acids that are newly made by ribosomes. Molecular chaperones, as their name suggests, are present during the protein-making process to ensure that newly made proteins behave normally and appropriately for their intended function. They do this by binding to the amino acid chain, thereby stabilizing it and facilitating proper folding. Another function that chaperones perform is to prevent folding errors and inappropriate aggregation, by recognizing incorrectly folded proteins and targeting them for repair or elimination. More information about molecular chaperones and proper protein folding can be found here.

Proteins folding incorrectly can lead to protein aggregation and disease. Moreover, there is a natural decline in a cell’s ability to continually make and fold protein correctly as one ages. As a result, in the study of HD, many scientists have focused their attention to the study of molecular chaperones and their role in responding to protein aggregation. TRiC is a molecular chaperone that has recently been found to be directly involved in regulating aggregation seen in proteins containing a long tract of glutamine amino acids, such as Htt.

What is TRiC and how is it involved in protein folding?^

TCP-1 ring complex, or TRiC, is a type of molecular chaperone that is composed of 8 different protein subunits, called CCT1, CCT2, all the way to CCT8 (CCT = chaperonin containing TCP-1). Together, these CCT subunits form a structure resembling two donuts stacked on top of each other.  Like other molecular chaperones, TRiC’s primary role in the cell is to act as a protein-folding machine: they bind to a specific set of newly made proteins and fold them into shapes that allow them to function correctly. This process occurs first with the unfolded protein entering and binding into the center of one of the rings in TRiC. TRiC then closes up the ring with the protein inside. During this time when the protein is enveloped in TRiC, protein folding occurs. After the protein is folded, TRiC can open up its ring again and eject the folded protein. In addition to huntingtin (Htt), some other proteins that are known to be regulated by TRiC include actin and tubulin, which are very important proteins that maintain the structural framework of the cell.

In order to identify molecular chaperones that are involved in proper folding of proteins with glutamine repeats, researchers looked at a roundworm often used in lab research. This 2006 study examined which proteins are important for minimizing aggregation of proteins with a polyglutamine expansion (“poly” means “many”, so “polyglutamine” means many glutamines). The researchers achieved this by using a roundworm that was genetically engineered to produce yellow fluorescent protein containing a polyglutamine expansion of 40 glutamine repeats. Then, they suppressed each gene in the roundworm one by one and looked for changes in the polyglutamine expanded fluorescent protein. The logic behind this experiment is that if a gene is important for preventing proteins with polyglutamine expansions, like Htt, from aggregating, then blocking that gene would increase the amount of protein aggregation. As you might have expected, one of the genes that the scientists identified through this experiment is the one that encodes the molecular chaperone TRiC.

Although roundworm models are easy to manipulate in the lab, it is much more relevant to study Htt aggregation in human cells. Consistent with the previous result, it has also been found that blocking TRiC in human cells will result in increased aggregation of proteins with an expanded glutamine repeat region.

How does TRiC actually suppress huntingtin aggregation?^

So far, we’ve discussed how TRiC is involved in folding newly formed proteins, but how does TRiC prevent already formed mutant huntingtin (mHtt) from aggregating?  Studies in this area of research first found that TRiC is able to directly bind to the Htt protein. This result aligns with what we previously knew about TRiC – that it acts as a molecular chaperone by assisting in protein folding. In particular, scientists have found, through several different experiments, that CCT1, one of the eight subunits forming TRiC, is especially important for binding to Htt. First, an in vivo study of the brain tissue of a transgenic mouse expressing mHtt revealed that TRiC and, specifically, CCT1 directly interacts with the Htt protein. This is very strong evidence indicating that TRiC regulates Htt activity in the brain in some way. Furthermore, by suppressing the CCT1 subunit alone, researchers still observe the same increase in Htt aggregation as when TRiC is suppressed. Even more significantly, expressing more CCT1 in cells can actually decrease the amount of Htt aggregation and toxicity in human cell culture models. All of these experiments suggest that TRiC can somehow control Htt aggregation through the CCT1 subunit.

Recent studies conducted between 2009 and 2013 from Judith Frydman’s lab at Stanford have shed light on how TRiC might control Htt aggregation through the CCT1 subunit.  First, researchers from the lab found that CCT1 binds to the Htt protein on a region of 17 amino acids, called N17, right next to the tract of glutamine repeats. This N17 region is significant because deleting this region in an Htt protein with 51 glutamine repeats appears to slow down the toxic protein aggregation process normally observed. In other words, the N17 region right next to the glutamine tract plays a role in speeding up the process of Htt protein aggregation.  Scientists also found that the N17 region of one Htt protein can bind to the N17 region and the glutamine tract of other Htt proteins.  Based on this evidence, they hypothesize that perhaps the N17 region of the Htt protein acts as a sticky end that other Htt proteins can attach onto and gradually form aggregates.

TRiC is another protein that can bind to the N17 region of Htt through its CCT1 subunit. Using advanced imaging techniques, the Stanford researchers found that when Htt and TRiC were put together, TRiC can either encapsulate single Htt proteins or small Htt aggregates in its ring chamber. It can also attach onto the tips of larger Htt aggregate fibers. From this experiment, the researchers concluded that TRiC can inhibit Htt aggregation perhaps by blocking the N17 region of Htt protein from endlessly sticking to other Htt proteins to form large, fibrous protein aggregates associated with neuron death. In the presence of TRiC, Htt will be maintained as a small single protein or small protein aggregates that are not toxic to the cell.

How can these findings about TRiC translate to an HD therapeutic?^

Research about TRiC is still in its preliminary stages, since much of the mechanisms behind TRiC’s ability to suppress huntingtin (Htt) aggregation remains to be understood. However, the fact that we do know TRiC and specifically its CCT1 subunit are potent inhibitors of aggregation is useful for researchers who are thinking of ways to stop the formation of dangerous aggregates that are toxic to brain cells.

A study published in 2012 by Sontag et al. took this knowledge and asked the following question: because cells that express more of the CCT1 subunit had less Htt protein aggregation, can this CCT1 subunit be applied as a drug and be directly delivered to brain cells? In order to answer this question, the researchers used cell models of HD and first showed that a modified fragment of CCT1 can penetrate the cell membrane and enter the cell to exert its effects. With regards to therapeutic benefits, the researchers observed that when higher doses of CCT1 were applied to rat cells engineered to express mHtt, there was a corresponding decrease in the amount of visible Htt aggregates formed. The scientists followed up with another experiment where they applied CCT1 to cells of HD mice that were removed from the striatum, the brain area most affected in HD.  They obtained perhaps an even more encouraging result: the cells receiving CCT1 treatment exhibited a higher survival rate compared to cells without treatment.  This suggests that CCT1 may exert therapeutic benefits for cells expressing mHtt beyond just suppressing protein aggregation.

Conclusion^

The above research is simply a small-scale experiment that demonstrates the potential that this line of research can have for HD therapeutics.  Evidently, there is a still a lot that we don’t know about how molecular chaperones like TRiC can suppress huntingtin (Htt) aggregation.  The experiments up to this point have all been performed with cells in laboratory settings, and these results will have to be verified in subsequent animal studies before any conclusions can be made about their actual therapeutic benefit.  However, studying TRiC has emphasized the importance of looking beyond the infamous glutamine repeats in Htt and looking at regions such as N17 to understand the mechanism of Htt aggregation and toxicity. Furthermore, the fact that direct protein delivery to the brain is already being done in mice for Parkinson’s disease research could lead the way for testing CCT1 in HD mouse models as well.

Further Reading^

Meyer AS, Gillespie JR, Walther D, Millet IS, Doniach S, Frydman J. (2003) Closing the Folding Chamber of the Eukaryotic Chaperonin Requires the Transition State of ATP Hydrolysis. Cell. 113(3): 369-381.

Behrands C, Langer CA, Boteva R, Böttcher UM, Stemp MJ, Schaffer G, Rao BR, Giese A, Kretzschmar H, Siegers K, Hartl FU. (2006). Chaperonin TRiC Promotes the Assembly of polyQ Expansion Proteins into Nontoxic Oligomers. Mol Cell. 23(6): 887-897.

Tam S, Geller R, Spiess C, Frydman J. (2006). The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 8(10): 1155-1162.

Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA, Frydman J. (2009) The Chaperonin TRiC Blocks a Huntingtin Sequence Element that promotes the Conformational Switch to Aggregation. Nat. Struct. Mol. Biol. 16(12): 1279-1285.

Shen K, Frydman J. (2013). The interplay between the chaperonin TRiC and N-terminal region of Huntingtin mediates Huntington’s Disease aggregation and pathogenesis. Protein Quality Control in Neurodegenerative Diseases. Eds. Morimoto  RI, Christen Y. 121-132.

Shahmoradian SH, Galaz-Montoya JG, Schmid MF, Cong Y, Ma B, Spiess C, Frydman J, Ludtke SJ, Chiu W. (2013). TRiC’s tricks inhibit huntingtin aggregation. eLife. 2:e00710.

Sontag EM, Joachimiak LA, Tan Z, Tomlinson A, Housman DE, Glabes CG, Potkin SG, Frydman J, Thompson LM. (2012). Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes. Proc. Natl. Acad. Sci. USA. 110(8): 3077-3082.

- J. Choi, 02-03-14