Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• ADAM17 (A disintegrin and metalloprotease 17) is a key enzyme regulating cell surface protein shedding.
• Cytoplasmic thioredoxin-1 (Trx-1) has been identified as a binding partner of ADAM17.
• The precise mechanism by which Trx-1 influences ADAM17 activity remains largely unknown.

Purpose of the Study:

• To elucidate the mechanism of ADAM17 regulation by its cytoplasmic partner, Trx-1.
• To determine the extent of Trx-1's influence on metalloproteinase ADAM17 activity.
• To investigate the roles of direct interaction and redox activity in Trx-1 mediated regulation.

Main Methods:

• Utilized discovery and targeted proteomic approaches.
• Performed cell-based assays, including synthetic peptide interactions and site-directed mutagenesis.
• Assessed Trx-1's reductive capacity and dimer formation using biochemical methods (gel filtration, mass spectrometry).
• Investigated the effects of reactive oxygen species inhibitors (NAC) and electron transport chain modulators (rotenone).

Main Results:

• Trx-1 negatively regulates ADAM17 through both direct and indirect mechanisms.
• The interaction interface between Trx-1 and ADAM17 is crucial for this regulation.
• Mutants of Trx-1 (K72A and C32/35S) differentially affected ADAM17 activity, highlighting the importance of both interaction and reductive capacity.
• The indirect effect is linked to Trx-1's diminished reductive capacity and altered cellular oxidant levels.
• Trx-1 K72A mutant exhibited reduced reductase activity due to increased dimer formation.

Conclusions:

• This study reveals a dual mechanism for Trx-1-dependent ADAM17 regulation: direct interaction and indirect redox modulation.
• The findings provide novel insights into the complex cross-talk between isomerases and mammalian metalloproteinases.
• Understanding this regulatory pathway is critical for deciphering ADAM17's role in various physiological and pathological processes.