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Human mitochondrial pyrophosphatase hPPA2: In vitro and in silico study of the factors affecting its function
Svetlana Kurilova1, Nataliya Vorobyeva1, Ekaterina Bezpalaya2
1A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia.
Abstract:
Biallelic mutations in a nuclear gene PPA2 in human encoding mitochondrial inorganic pyrophosphatase hPPA2 cause mitochondrial disfunctions leading to severe cardiac pathology. This protein catalyzes a Mg2+-dependent hydrolysis of pyrophosphate, a by-product of many biosynthetic reactions, thereby providing a thermodynamic pull for these reactions. In order to better understand molecular mechanisms of mitochondrial disfunction caused by mutations in PPA2 gene, detailed characterization of a metabolic role of hPPA2 is required. In this work, we study the in vitro effects of a panel of metabolites, as well as other factors, on the recombinant hPPA2 activity. This study is complemented with the in silico assessment of possible mechanisms of observed patterns. We demonstrate that at Mg2+ concentrations typical for the mitochondrial matrix hPPA2 works at under-saturation and therefore the regulatory factors changing Mg2+ concentration will have a significant impact on hPPA2 activity. We also demonstrate that hPPA2 activity is regulated by a redox state of cysteine residues. Mass-spectrometry analysis reveals four Cys residues modified by 4-hydroxy-Hg-benzoate, as well as an SS bridge formation within an hPPA2 monomer. Finally, we found that selected metabolites including intermediates of central metabolism affect hPPA2 activity. In silico analysis of hPPA2 allows a structural insight into the observed properties. Of a special interest is an W-loop, unique for PPA2 proteins from animals and shared with acidocalcisomal soluble pyrophosphatases from kinetoplastids, that may be involved in interaction with small-molecule effectors.
Insights
Mutations in the PPA2 gene cause cardiac issues by affecting mitochondrial inorganic pyrophosphatase (hPPA2). This study reveals hPPA2 activity is regulated by Mg2+ levels, redox state, and metabolites, offering insights into mitochondrial dysfunction.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Biallelic mutations in the nuclear gene PPA2 lead to mitochondrial dysfunction and severe cardiac pathology.
- The protein hPPA2 (human inorganic pyrophosphatase) hydrolyzes pyrophosphate, crucial for biosynthetic reactions.
- Understanding hPPA2's metabolic role is key to elucidating PPA2-mutation-related mitochondrial dysfunction.
Purpose of the Study:
- To characterize the in vitro effects of metabolites and other factors on recombinant hPPA2 activity.
- To investigate the regulatory mechanisms of hPPA2, including Mg2+ dependence and redox sensitivity.
- To gain structural insights into hPPA2 function using in silico analysis.
Main Methods:
- In vitro enzymatic assays with recombinant hPPA2.
- Mass-spectrometry analysis to identify cysteine modifications and disulfide bridges.
- In silico structural modeling and analysis.
Main Results:
- hPPA2 operates under-saturated at typical mitochondrial Mg2+ concentrations, making it sensitive to Mg2+ level fluctuations.
- hPPA2 activity is redox-regulated, with cysteine residues modified and involved in disulfide bond formation.
- Metabolites, including central metabolism intermediates, were found to modulate hPPA2 activity.
- In silico analysis highlighted a unique W-loop potentially involved in effector interactions.
Conclusions:
- Mitochondrial inorganic pyrophosphatase (hPPA2) activity is finely tuned by Mg2+, redox state, and metabolites.
- These regulatory mechanisms are critical for understanding mitochondrial dysfunction in PPA2-related cardiac pathologies.
- Structural features like the W-loop may mediate interactions with small molecules, offering therapeutic targets.
