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Cellular protein damage control: interactomic analyses of MOAG-4 in C. elegans

Final Report Summary - AGGREGATION (Cellular protein damage control: interactomic analyses of MOAG-4 in C. elegans)


The organization of proteins into amyloid fibrils is wide spread in nature from bacteria to human. Several functions have been described for amyloids, ranging from structural to regulatory roles. However, the formation of amyloid fibrils in the brain and the toxicity generated by the protein aggregation process have been related to several age-related neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases. The cellular process of aggregation, its regulation, and the toxicity generated are not completely understood. Small models have been traditionally used to gain insight into this mechanism and several neurodegenerative diseases related to protein misfolding and aggregation have been modelled in the nematode C. elegans. With the use of the C. elegans model for Huntington’s disease, and by mutagenesis screen, we identified a positive regulator of aggregation, called MOAG-4.
In worms, MOAG-4 was shown to be able to promote the aggregation and toxicity of three disease proteins PolyQ, amyloid-ß and α-synluclein. The role of MOAG-4 is evolutionarily conserved in the human orthologs SERF1A and SERF2. MOAG-4/SERFs appear to regulate age-related proteotoxicity through a previously unexplored pathway. However, it has been demonstrated in vitro that SERF1A is able to specifically promote the aggregation of amyloidogenic proteins, while not being able to do the same with non-amyloidogenic proteins.


The main aim of this project was to unravel the pathway through which MOAG-4/SERF enhance aggregation of several neurodegenerative disease proteins. In this context, we have focused on the endogenous role of MOAG-4/SERF under non-disease conditions and on understanding how it is related with protein aggregation. In order to do that, we have studied the interaction of MOAG-4/SERF with other proteins in the cell and how these interactions can explain their role in protein aggregation.
Summing up, our objectives were on a first moment to [1] Identify MOAG-4/SERF interacting proteins; [2] to determine the role of the MOAG-4/SERF interactors in protein aggregation. On a second moment, we have [3] combined human cells and C.elegans in order to quantify the evolutionary conservation of our findings.


I. Identification of MOAG-4/SERF interacting proteins.

We used SERF1A and SERF2 as bait in a Yeast 2 Hybrid (Y2H) experiment to screen for possible interacting proteins in the human proteome. We replicated the screen four times for two different genetic reporters. After applying a restrictive cutoff, we obtained a list of 40 proteins interacting with SERFs with high affinity. We used different bioinformatic analysis tools to find common biological functions or proteins domains which could give us any hint to understand their relationship with SERFs and protein aggregation. However, after that analysis we found that the SERF-interacting proteins were functionally unrelated.
Knowing that SERF can drive amyloid formation of a variety of functionally unrelated proteins, we next investigated the possibility of the interactors as putative SERFs endogenous substrates. We first performed an in silico prediction of their propensity to form amyloids and we determined that most of them have at least one peptide with predicted amyloidogenic properties.

II. Determination of the role of the MOAG-4/SERF interactors in protein aggregation.

In order to address whether the SERF interactors were endogenous substrates, we analyzed the capacity of these proteins to form aggregates in vivo. For that, we expressed all 40 proteins fused to GFP in cells, which allowed us to visualize the formation of inclusions. The latter was followed by the analysis of their SDS-insolubility by subfractionation and western blot. As a result, we found that 30 out of the interactors became SDS insoluble, similar to known amyloid forming proteins.
To address the question whether the aggregation of the SERF interactors was SERF dependent and, therefore, to establish whether they were substrates, we repeated the same experiments in WT and SERF-double-knock-out mutant cell lines. We found that for 20 out of 40 proteins, the aggregation was altered in the mutant background, suggesting that the interactors are SERFs substrates, and that there is a role for SERFs in the cell as general regulator of aggregation for other proteins than the known disease proteins.
Since MOAG-4/SERFs function was firstly identified in C. elegans we wanted to study whether these new findings could also be found in worms and whether the new described role of SERFs is evolutionary conserved. With this aim in mind, we identified 21 orthologues in C. elegans for the SERF interactors, and we got the strains expressing the GFP-tagged version of the proteins for 8 of them. Since a fluorescent signal could only be found for 2 out of those 8, we added into the analysis another previously found protein by using an immunoprecipitation approach in worms. In this way, we reproduced with these worm strains the experiments previously described for cells, and we found that two of the analyzed proteins formed SDS-insoluble inclusions during ageing and that this aggregation was reduced in the moag-4 mutant strains. These results confirm the same role for MOAG-4 in worms as for SERFs in human cells.
In addition, when the expression of the genes for these two proteins was knocked down by RNAi in the worm model for Huntington we observed an increase in the polyQ aggregation, suggesting a competition between the endogenous and exogenous substrates for MOAG-4.

During this fellowship, we have obtained promising results presenting MOAG-4/SERFs as a general regulator of protein aggregation.
In order to further explore the potential of SERF as a target to treat protein aggregation in neurodegenerative diseases, we decided to continue this project beyond the Marie Curie Fellowship, and look for further evidence to support our hypothesis.