hyP5 · Adopting orphan pumps: Structural and functional characterization of P5-ATPases
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2021-09-01 → 2023-08-31
- EU contribution
- €219,312
- Participants
- 1
- Scheme
- MSCA-IF
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Results in brief
Adopting orphan pumps: Structural and functional characterization of P5-ATPases
The aim of this project was the structural and functional analysis of yeast and human P5 ATPases. P5 ATPases belong to the P-type ATPase superfamily of essential membrane transport proteins and are conserved in all eukaryotes. This class of proteins utilizes ATP to transport specific substrates across membranes. The substrates that are being transported by P-type ATPases are very diverse, ranging from small Metal-ions to large substrates such as lipids. P5 ATPases can be further divided into P5A and P5B ATPases. It has been shown that the yeast P5A Spf1p extracts single-spanning transmembrane proteins out of the ER membrane, while members of P5B subclass transport polyamines. This project was focusing on investigating Spf1p, the yeast P5A ATPase and ATP13A2, the human P5B ATPase. It has been found that mutations in human P5 ATPases cause severe neurodegenerative diseases, such as familial early-onset parkinsonism and autism/ language disorders. To better understand how mutations alter the protein and influence the substrate transport, it is important to obtain structural information on the protein. Protein structures help to better understand the function of the protein and to design potential drugs. The specific objectives of this project were to 1) functionally characterize P5 ATPases to better understand their transport mechanism, 2) to obtain structures of different states during the catalytic cycle and co-structures with substrates/ inhibitors to help better targeting drug development, and 3) to analyze the interaction network as malfunction of this class of proteins have been shown to have a broad phenotype and therefore influence many downstream processes.
Data: CORDIS, © European Union
Project objective
P5-ATPases are conserved in all eukaryotes and malfunctions in human are associated with severe neurological diseases, such as familial early-onset parkinsonism and autism/language disorders, and with phenotypical traits in yeasts. They belong to the P-type ATPase superfamily, which encompass a range of essential membrane transporters for ions and lipids. Ion pumps such as Na,K-ATPase and Ca2+-ATPase have been studied in great detail during the last decades. However, astonishingly little is known about the P5-ATPases and their actual function, despite their physiological importance in all eukaryotes.The current proposal focuses on substrate identification and structural characterization of P5-ATPases, as well as investigations of their cellular interaction network. Human P5-ATPases (ATP13A1 through 5, ATP13A2 also known as PARK9) and the yeast orthologues Spf1p and Ypk9p will be subjects of this study. Target proteins will be expressed in their native host (yeast or HEK cells) and subsequently purified and used for activity assays, structural studies, and identification of interaction partners. Native mass spectrometry will identify bound substrates and cofactors, and activity studies will elucidate structure-function relationships. 3D-structures obtained by single-particle cryo-electron microscopy (cryo-EM) and/or X-ray crystallography will reveal catalytic mechanisms and mutational effects. Structural and functional characterization of P5-ATPases can therefore serve as a basis for understanding molecular mechanisms of e.g. neurodegenerative and cognitive disorders and guide novel strategies in disease treatments and drug discovery.Using my profound experience from my PhD with crystallography of biotechnologically relevant proteins, I wish to pursue a postdoc focused on membrane proteins with a strong potential in molecular medicine and to expand my knowledge of methods in structural biology and molecular cell biology, in particular cryo-EM.
Original text from CORDIS.
Participants
- AARHUS UNIVERSITET · Aarhus CCoordinatorDenmark
Links
Data: CORDIS, © European Union