A chemical biology approach to elucidate the function of protein pyrophosphorylation in the nucleus and nucleolus

• Applicant: Professorin Dr. Dorothea Fiedler
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469186007

The goal of this proposal is to elucidate the role of an unusual signaling mechanism termed protein pyrophosphorylation. Pyrophosphorylation is an understudied protein modification, thought to be mediated by inositol pyrophosphate messengers. While the inositol pyrophosphates have been linked to numerous important cellular processes, the function of protein pyrophosphorylation remains poorly characterized because suitable molecular tools and detection methods are lacking. Here, we will implement an interdisciplinary approach, including chemical synthesis, peptide synthesis, protein semi-synthesis, phospho- and pyrophosphoproteomics, isotopic labeling, proximity labeling, and chromatin biology, to ignite this underdeveloped field. Our studies will be initiated by global annotation of pyrophosphorylation sites, applying a tailored mass spectrometry (MS) method. MS analysis will also uncover the dynamics of these sites and determine which stimuli this modification responds to. Using proximity labeling in the nucleus, we will then explore how pyrophosphorylation can serve as a specific signal, by identifying proteins which specifically interact with pyrophosphoserine – so called “pyrophosphoserine readers”. And lastly, we will unravel how pyrophosphorylation can alter protein function and structure of two nuclear proteins, SUB1 (activated RNA polymerase II transcriptional coactivator p15 , also termed PC4) and HDAC2 (histone deacetylase 2). We will develop methodology to generate complex PP-Ser containing peptides, which will then be incorporated into full-length SUB1 and HDAC2 using different ligation strategies. Illuminating all of these different aspects on the function and regulation by protein pyrophosphorylation is essential for providing an integrated view at the cellular, and ultimately, the organismal level.While protein pyrophosphorylation has not been linked to specific phenotypes so far, our preliminary MS data point to a role in nuclear signaling and nucleolar biology. Therefore, our current research proposal will focus on the putative function of pyrophosphorylation in nuclear responses, regulating transcription and chromatin remodeling. By making our methods for the detection and characterization of pyrophosphorylation accessible to researchers across fields, elucidation of the relevance of this modification in various biological contexts will be greatly accelerated.

• DFG Programme: Research Grants
• International Connection: China
• Partner Organisation: National Natural Science Foundation of China
• Cooperation Partner: Professor Mingxuan Wu