Periodic Reporting for period 3 - RibiTool (Ribitol-phosphate: chemical tools to probe the biology of a unique mammalian carbohydrate)
Reporting period: 2023-02-01 to 2024-07-31
Sugars are commonly known for the nutritional role they play in our bodies. Aside from providing much-needed energy that fuels our cells, however, sugars - also referred to as carbohydrates - are important for many other biological processes including as structural components and for communication between cells and their surroundings. This project focuses on a specific type of carbohydrate that helps muscle cells to make vital connections with the surrounding tissue. It is well established that genetic variations affecting the cell’s ability to produce these carbohydrates leads to a group of inherited muscular dystrophies termed alpha-dystroglycanopathies. Yet many questions remain about exactly how cells control the production of these carbohydrates and how various genetic changes lead to the wide spectrum of symptoms and disease outcomes that has been observed in patients suffering from alpha-dystroglycanopathies.
The ERC-funded research programme aims to answer some of these questions by building a more comprehensive understanding of the cellular processes that are at the basis of these disorders. It will do so by exploring a completely new, multi-disciplinary approach that combines elements of chemistry and biological research to deliver bespoke technologies and insights that will help us better understand the molecular basis of disease.
The overall objectives include:
1. Developing new chemical technologies to detect and characterise the carbohydrate of interest in its natural environment.
2. Establishing methods for studying individual components of the cell’s machinery responsible for producing the carbohydrate of interest.
3. Developing new research tools with which to further study the same components as those under objective (2) in their natural environment.
4. Bringing together all technologies and methods described above for studying the consequences of disease-causing genetic changes from various different angles.
It is anticipated that the project outcomes will generate new ideas for putative therapeutic strategies and targets while also establishing robust diagnostic methodologies to evaluate the effectiveness of such strategies.
The ERC-funded research programme aims to answer some of these questions by building a more comprehensive understanding of the cellular processes that are at the basis of these disorders. It will do so by exploring a completely new, multi-disciplinary approach that combines elements of chemistry and biological research to deliver bespoke technologies and insights that will help us better understand the molecular basis of disease.
The overall objectives include:
1. Developing new chemical technologies to detect and characterise the carbohydrate of interest in its natural environment.
2. Establishing methods for studying individual components of the cell’s machinery responsible for producing the carbohydrate of interest.
3. Developing new research tools with which to further study the same components as those under objective (2) in their natural environment.
4. Bringing together all technologies and methods described above for studying the consequences of disease-causing genetic changes from various different angles.
It is anticipated that the project outcomes will generate new ideas for putative therapeutic strategies and targets while also establishing robust diagnostic methodologies to evaluate the effectiveness of such strategies.
Objective 1: we have designed a set of carbohydrates derived from the natural sugar but equipped with unnatural functionality that acts as an identification tag when studying the sugar in a biological context. The concept of using unnatural sugars to study biological processes has been published in Biochemical Society Transactions 2021. We have developed a new chemical methodology for the synthesis of a key element of these unnatural sugar derivatives - a biologically cleavable phosphotriester group. We have published this work in the first instance as a preprint on ChemRxiv and then submitted it for publication in Chemical Science, where it is currently under revision. Furthermore, using this methodology, we have produced 11 of the unnatural sugars and prepared them for biological studies.
Objective 2: three human proteins that are involved in the production of the carbohydrate of interest within cells has been selected for in-depth characterisation by biochemical analysis and 3-D structure analysis. This requires the proteins to be produced and isolated from other cellular components. We have started the production of these target proteins and currently have one of them (ISPD) ready for further analysis, while work on one of the other two (FKTN) is ongoing. We are also working on a set of bacterial proteins (TarI, TarK, TarL) that are similar to the human targets to facilitate method development and to enable comparisons between the two families. Production of these targets is well underway, and we have established a working pipeline for the successful isolation of two of these targets. Finally, we have begun biochemical and crystallographic analysis of ISPD and TarK (unfortunately no crystals have formed so far).
Objective 3: using a similar approach to that described in objective 1, we have designed two different sets of unnatural carbohydrates as research tools to help us study not the sugar itself, but the cellular components responsible for producing them (namely, the same proteins as those in objective 2). Production is ongoing and initial proof of concept analyses have revealed that these sugars do indeed engage with the anticipated target protein as expected. Further derivatives are being designed and methods for their production developed. A review on the topic was published in Molecules 2021.
Objective 4: a cellular assay has been established for the testing of the unnatural sugars from objective 1. Initial tests have revealed that 3 of these reproducibly label a number of cellular proteins. Further work will be needed to establish the identity of these proteins and their glycans. The tools developed in objective 3 will initially be tested on the isolated proteins described in objective 2, before applying them to the cellular assay. Work has also begun to generate a fluorescently tagged version of one of the target proteins (FKTN) and mutants carrying a selection of known disease-causing variations to enable studies into the consequences of these mutations.
Objective 2: three human proteins that are involved in the production of the carbohydrate of interest within cells has been selected for in-depth characterisation by biochemical analysis and 3-D structure analysis. This requires the proteins to be produced and isolated from other cellular components. We have started the production of these target proteins and currently have one of them (ISPD) ready for further analysis, while work on one of the other two (FKTN) is ongoing. We are also working on a set of bacterial proteins (TarI, TarK, TarL) that are similar to the human targets to facilitate method development and to enable comparisons between the two families. Production of these targets is well underway, and we have established a working pipeline for the successful isolation of two of these targets. Finally, we have begun biochemical and crystallographic analysis of ISPD and TarK (unfortunately no crystals have formed so far).
Objective 3: using a similar approach to that described in objective 1, we have designed two different sets of unnatural carbohydrates as research tools to help us study not the sugar itself, but the cellular components responsible for producing them (namely, the same proteins as those in objective 2). Production is ongoing and initial proof of concept analyses have revealed that these sugars do indeed engage with the anticipated target protein as expected. Further derivatives are being designed and methods for their production developed. A review on the topic was published in Molecules 2021.
Objective 4: a cellular assay has been established for the testing of the unnatural sugars from objective 1. Initial tests have revealed that 3 of these reproducibly label a number of cellular proteins. Further work will be needed to establish the identity of these proteins and their glycans. The tools developed in objective 3 will initially be tested on the isolated proteins described in objective 2, before applying them to the cellular assay. Work has also begun to generate a fluorescently tagged version of one of the target proteins (FKTN) and mutants carrying a selection of known disease-causing variations to enable studies into the consequences of these mutations.
All of the produced unnatural sugars are conceptually new and, aside from their significance to the biological studies described here, are of interest to the chemical community because of the new chemical methodologies that have been developed to produce them. In particular, one step of the production process presents a significant advance to the state of the art in the field of carbohydrate chemistry and has been published (as a preprint, soon also in peer-reviewed literature) as a standalone methodological research paper. We expect to be able to finish the production of all remaining sugars from objective 1 and several of those from objective 3 (noting a subset of the designed sugars have proven unstable and can not be isolated) before the end of the project. We will also test all of the produced sugars using the proteins produced in objective 2, if available, and the cellular assay developed in objective 4. Having identified successful research tools, we will then proceed with follow-up experiments in which the successful tools will be used for more in-depth characterisation of the carbohydrate of interest, the cell’s machinery responsible for producing it, and the consequences of genetic variations that cause muscular dystrophy. These biological studies are expected to take up the majority of time in the remaining part of the project.
A number of the proteins described in objective 2 have not previously been subjected to 3-D structural studies. Being able to produce 3-D structures, and use this to better understand how the proteins work and contribute to disease and to inform the design of further research tools, will have a significant impact on the biological and biomedical research communities. During the remainder of the project, we will aim for complete production and isolation of all three human target proteins. As some of these proteins have proven extremely challenging to produce, we will also continue our work on the more tractable bacterial proteins, which will function as models for assay design and probe testing. We will then develop assays to test the proteins’ properties in vitro (to complement the cellular studies under objective 4) and carry on with studies on the effects of disease-causing variations.
A number of the proteins described in objective 2 have not previously been subjected to 3-D structural studies. Being able to produce 3-D structures, and use this to better understand how the proteins work and contribute to disease and to inform the design of further research tools, will have a significant impact on the biological and biomedical research communities. During the remainder of the project, we will aim for complete production and isolation of all three human target proteins. As some of these proteins have proven extremely challenging to produce, we will also continue our work on the more tractable bacterial proteins, which will function as models for assay design and probe testing. We will then develop assays to test the proteins’ properties in vitro (to complement the cellular studies under objective 4) and carry on with studies on the effects of disease-causing variations.