Microglia are macrophage-like cells resident within the central nervous system, responsible for the immune response. Activated microglia secrete inflammatory cytokines and lysosomal peptidases, including cysteine peptidases. Overactivation and dysregulation of microglia might result in neuron damage and the development of neurodegenerative diseases. An endogenous inhibitor of cysteine proteases is cystatin F, which belongs to the type 2 cystatins and is expressed mainly in immune cells. Cystatin F is synthesized as an inactive dimer that requires proteolytic cleavage and monomerization for its activation. Our study aimed to determine cystatin F’s intracellular localization and its inhibitory targets in human microglia using immunocytochemistry. Western blot and measurements of the activity of the cysteine cathepsins and legumain were used to define cystatin F’s role on its targets. We confirmed the presence of legumain in the lysosomes and a strong colocalization of cystatin F with legumain and cathepsin L, while the colocalization with cathepsin C was weaker. Additionally, we also proved colocalization of legumain and cathepsin L. Internalized wild-type cystatin F reduced the processing of the single-chain form of cathepsin L to the two-chain form and activation of cathepsin C and legumain from precursor forms. The addition of cystatin F significantly reduced the activity of cathepsins C, L and legumain. Cystatin F with a mutated binding site for legumain showed no effect on the processing of cathepsin C and legumain, while a decrease in the processing of the single-chain form of cathepsin L to the two-chain was detected. Mutated cystatin F did not affect the activity of cathepsins C and L, however, this could be a consequence of too big differences in variances. Our results show that, unlike cytotoxic lymphocytes, the main targets for cystatin F in human microglia are cathepsin L and legumain and also cathepsin C. That can have an impact on the phagocytic activity of microglia.
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