Date: 17th March 2022
The tumour suppressor p53, is a key player in the human body’s defence against cancer. It plays a central role in the control of cell proliferation, apoptosis and genomic stability and helps discover and prevent genetic mutation in cells, which could turn cancerous. Mutations that inactivate p53 are found in up to 60% of all human cancers therefore, restoring its function has long been an attractive therapeutic strategy. However, p53 is poorly expressed and conformationally unstable meaning it quickly breakdowns in cells hindering its use as a cancer therapeutic. Now, researchers have found an innovative way of stabilising p53 and making it more potent, by engineering a spider silk domain to the p53 protein they have created a fusion protein with increased expression and stability, capable of killing cancer cells.
Intrinsically disordered proteins (IDPs) are characterised by the lack of a stable tertiary structure under physiological conditions. Many key tumour suppressors have large intrinsically disordered domains (IDDs), and their ability to play roles in a variety of essential cellular processes makes them attractive targets for therapeutic approaches. One such example is p53 however, its low expression levels and poor conformational stability has to date hampered drug design efforts.
Now, researchers at Karolinska Institutet, Sweden, led by Michael Landreh, were inspired by how nature creates stable proteins and have used spider silk protein to stabilise p53. The spider silk-p53 fusion protein adopted a compact state, was biologically active and was more stable than native p53. It was expressed in cells in large quantities and the new protein was capable of killing cancer cells.
The team started by asking whether p53 could be pulled back from “the edge of solubility” by fusing it to a protein that was highly expressed and particularly stable against aggregation. They used a conserved N-terminal (NT) domain of spider silk, which the team had previously shown when mutated into a non-dimerising version (NT∗) enabled production of aggregation-prone peptides by increasing expression while suppressing self-assembly. They fused NT∗ to the N-terminal of p53, and showed it increased translation efficiency in vitro, but that the NT∗ domain did not alter the conformation or oligomerisation of the p53 moiety. The presence of NT∗ did however, induce compaction of p53’s intrinsically disordered transactivation domains.
Based on this observation that the fusion protein was now expressed in a compact folded state, the team asked whether it would retain its functionality in human cancer cells. The majority of the protein could be detected in the nucleus of the cells (as was wild type p53) and did not form aggregates, and was able to specifically induce targets related to cell-cycle arrest and apoptosis.
Whilst, NT∗ was able to unblock translation of p53, the team wanted to assess whether it could confer these properties to other proteins. As with disordered p53, the spider silk domain could also promote translation of B-Raf, which is also disordered, however no enhancement was observed when applied to the folded K-Ras. This suggested that NT∗ could efficiently increase protein translation by reducing N-terminal disorder.
The team here have shown that a spider silk domain can boost translation of p53 by inducing a compact conformation of its intrinsically disordered N-terminal domain. This open the doors for promising new avenues of cancer therapy, by stabilising cellular p53.
The team are hopeful that the strategy can be adapted to target a wide range of partially disordered proteins. Indeed, the expression of B-Ras, a partially disordered cancer target, was similarly enhanced by NT∗. This method should facilitate structural investigations of particularly challenging systems.
The researchers now plan to study the protein’s structure in detail and how its different regions interact to prevent cancer. Their long term goal is to develop an mRNA-based cancer vaccine, and this system gives them the tools for the initial discovery work.
In situ vaccination is a promising strategy for cancer immunotherapy however, there are currently only two FDA-approved vaccines that prevent cancer and only one that can treat existing cancer. Recent work using lipidoid nanoparticles (LNPs) to enhance the effect of in situ vaccination by promoting cross-presentation of tumour antigens and activating the immune response via the STING pathway, resulted in ablation of tumours and induced immune memory. AI-powered cancer vaccines have also entered clinical trials following the success of preclinical studies. Other strategies being explored are the use of implantable blood clot cancer vaccine.
However, as we have seen with rapid development of mRNA COVID-19 vaccines, with for example BioNTech’s vaccine being the first mRNA vaccine to ever be approved for use, these are highly effective and now represent a valuable and agile method for vaccine production. The ability to stabilise tumour suppressor proteins such as p53, and use the lesson learnt from this, will hopefully accelerate a new generation of cancer vaccines for clinical use in the near future.
For more information please see the press release at the Karolinska Institutet
Kaldmäe, M., Vosselman, T., Zhong, X., Lama, D., Chen, G., Saluri, M., Kronqvist, N., Siau, J.W., Ng, A.S., Ghadessy, F.J., et al. A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder. Structure.