Periodic Reporting for period 4 - MetAGEn (Metabolic and Genetic Regulation of Ageing)
Reporting period: 2020-02-01 to 2020-07-31
Seeking for ways to improve protein quality, I have identified a novel longevity pathway in Caenorhabditis elegans. In a forward genetic screen, I found a link between metabolites in the hexosamine pathway and cellular protein quality control. Hexosamine pathway activation extends C. elegans lifespan, suggesting modulation of ageing by endogenous molecules.
In a first step, I am exploring the mechanism by which hexosamine metabolites improve protein quality control in mammals, using cultured mammalian cells and a mouse model for neurodegeneration. Preliminary data show that hexosamine pathway metabolites enhance proteolytic capacity in cells and reduce protein aggregation, suggesting conservation. Second, I am investigating molecular mechanisms that activate the hexosamine pathway to promote protein homeostasis and counter ageing. Third, I am performing a direct forward genetic screen for modulators of ageing in C. elegans. For the first time, mutagenesis and next generation sequencing can be paired in forward genetic screens to interrogate the whole genome for lifespan-extending mutations in a truly unbiased manner. This innovative approach has the potential to reveal novel modulators of the ageing process.
Taken together, this work aims to understand molecular mechanisms that maintain cellular homeostasis to slow the ageing process, and to develop a new technology to identify yet unknown genetic modulators of ageing.
Conclusion of the Action:
1. I have investigated how the hexosamine pathway modulates cellular protein quality control in mammalian cells. Our published work (Horn et al., 2020 iScience) shows that hexosamine pathway activation is a trigger for a conserved stress response pathway, the integrated stress response (ISR). A mild ISR activation that we observed both in C. elegans and in mammalian cells, activated autophagy that was required to clear toxic protein aggregates.
2. Through biochemical and structural biology approaches we have delineated the molecular regulatory mechanism of GFAT-1 the key enzyme of the hexosamine pathway (Ruegenberg et al., 2020 Nature Commun).
3. We expanded, beyond the initial scope of the project, our research to genetic screens in haploid cells which enable high-resolution mechanistical insights in to protein function (Horn et al., 2018 Oncotarget; Allmeroth et al., 2020 Leukemia). This work has also revealed new insights into the regulation of the hexosamine pathway.
4. We performed a forward longevity screen in C. elegans, as outlined in the initial proposal, and have now identified many new longevity loci, one of which revealed a new function of the ISR in longevity (Derisbourg et al., 2020 bioRxiv)
II. Molecular characterization of GFAT-1. I will investigate the biochemical mechanism of GFAT-1 gof through enzyme kinetic assays and an unbiased search for interaction partners. We have now successfully purified recombinant human GFAT-1 and have established a number of activity assays. This work has revealed the mechanism for the GFAT-1 gain-of-function. We have completed this work and found multiple independent gain-of-function mechanisms of GFAT-1 that are all conserved in the human protein. Loss of feedback inhibition is a key gain-of-function mechanism.
III. Unbiased direct forward genetic screen for longevity genes in C. elegans. I will perform the first forward genetic screen that uses longevity as a primary readout. We have now generated more than 100 long-lived C. elegans mutants and have completed next-generation sequencing for all of these within reporting period 1. Analysis of this rich and unique data set has revealed a number of candidates for novel longevity genes are we are in the process of confirming these using the CRISPR/Cas9 technology. Next, we will prioritize on the most interesting mutants and will devise a publication strategy. We have selected a group of new longevity mutants that control protein biosynthesis and the ISR and, through this work, we identified a novel aspect in the regulation of selective protein biosynthesis that directly affects aging.
In a project that goes beyond the original proposal, we have adapted haploid mouse embryonic stem cells for mutagenesis screens and implemented an approach that enables screens at amino acid resolution, providing direct insights into protein function. We have applied this in my team as well as in numerous collaborations and have founded a start-up company based on this technology and with help from an ERC PoC grant (ACUSLABS).
II. GFAT-1 activation occurs through loss of feedback inhibition.
III. Unbiased direct forward genetic screens for longevity genes in C. elegans have indicated an unexpected role of the integrated stress response in longevity.