Antimicrobial peptide LL-37 is both a substrate of Cathepsins S and K and a selective inhibitor of Cathepsin L

Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family

A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity