title: Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions.
abstract: BACKGROUND
  Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells.
  
  
  RESULTS
  The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions.
  
  
  CONCLUSIONS
  The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, Campus Ring 6, Research II, Bremen, Germany. sjordans@jacobs-alumni.de
authors.forename: Silvia
authors.initials: S
authors.lastname: Jordans
authors.forename: Sasa
authors.initials: S
authors.lastname: Jenko-Kokalj
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Kühl
authors.forename: Sofia
authors.initials: S
authors.lastname: Tedelind
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sendt
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.forename: Dusan
authors.initials: D
authors.lastname: Turk
authors.forename: Klaudia
authors.initials: K
authors.lastname: Brix
mesh_terms: D002403:Cathepsins / Q000378:metabolism*; D002451:Cell Compartmentation; D003545:Cysteine / Q000378:metabolism; D005110:Extracellular Space; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008247:Lysosomes / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013954:Thyroglobulin / Q000032:analysis; D013961:Thyroid Gland / Q000201:enzymology* / Q000473:pathology
chemical_list: D010446:Peptide Fragments; D013954:Thyroglobulin; D002403:Cathepsins; D003545:Cysteine