DOMAIN-PHOS · Towards large-scale functional characterization of protein phosphorylation

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Project objective

In recent decades, phosphoproteomics has greatly advanced our ability to quantify protein phosphorylation. However, characterizing the functional consequences of these phosphorylation events remains challenging, with only ~6% of recorded events having an associated function. This project aims to bridge this gap by developing a high-throughput method for characterizing biochemical functions of phospho-events.The proposed approach, supported by preliminary data, leverages the fact that some protein domains recognize phosphorylation of other proteins, thereby defining the function of both the binder and the phospho-substrate. For example, 37 human proteins contain the phospho-binding Forkhead-Associated (FHA) domain, each with a slightly altered sequence influencing substrate choice. FHA proteins act in various processes, including DNA damage response and cytoskeletal movements, areas of expertise for the applicant.The project involves GFP-tagging all human FHA domains for use in affinity pulldowns, followed by mass spectrometry for comparative analysis of phospho-substrates. For validation, we will perform large-scale reciprocal phosphopeptide pulldowns, a technique developed in the host lab. The combination of the applicant's expertise and the host lab's methodology creates a strong synergy. To understand structure-function relations, we will model interactions of shortlisted FHA:phospho-substrate pairs using AlphaFold3. Additionally, we will conduct phenome-wide association studies to find links between mutations in interaction pairs and disease states, including the functional follow-up on disease-relevant findings.The appeal of this approach extends beyond expanding FHA domain biology, as it has the potential to generalize to other post-translational modification- (PTM-) binding domains. This method could start a field describing PTM-associated functions at scale, enabling easier causative linking of protein function, mutation, and disease.

Original text from CORDIS.

Participants

• KOBENHAVNS UNIVERSITET · KOBENHAVNCoordinatorDenmark

Links

• View on CORDIS
• DOI: 10.3030/101209788
• https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e52c709008&appId=PPGMS