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Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating.

Rosanna P Baker1, Siniša Urban1

  • 1Howard Hughes Medical Institute, Department of Molecular Biology &Genetics, Johns Hopkins University School of Medicine, Room 507 PCTB, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.

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Summary

Calcium ions regulate intramembrane proteases, specifically rhomboid-4, by controlling substrate gating. This discovery offers new insights into cell signaling and disease-related protease functions.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Intramembrane proteases are crucial for cell signaling and implicated in diseases.
  • Direct regulation mechanisms for these membrane-bound enzymes remain largely unknown.

Purpose of the Study:

  • To investigate the direct regulation of intramembrane proteases, focusing on a rhomboid protease with calcium-binding EF-hands.
  • To elucidate the role of calcium in rhomboid protease activity and substrate processing.

Main Methods:

  • Characterization of a calcium-binding rhomboid protease (rhomboid-4) in Drosophila cells.
  • Purification and liposome reconstitution of rhomboid-4 to study calcium-dependent proteolysis.
  • Analysis of EF-hand deletions and mutations in cytoplasmic loops to identify regulatory regions.

Main Results:

  • Calcium strongly stimulates proteolysis by rhomboid-4 in both cellular and reconstituted systems.
  • EF-hand domains are essential for preventing premature proteolysis, while cytoplasmic loops mediate calcium-induced stimulation.
  • Regulation occurs via calcium-mediated substrate gating, not dimerization or substrate interaction.
  • Substrates cleaved outside the membrane lose regulatory capacity, suggesting gating is specific to intramembrane proteolysis.

Conclusions:

  • Substrate gating is an evolved regulatory mechanism for intramembrane proteolysis, not essential for catalysis itself.
  • Calcium acts as a direct regulator of rhomboid protease activity through substrate gating.
  • These findings open new avenues for studying rhomboid protease function and upstream regulatory inputs.