An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L‐like cysteine proteases

Abstract

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

“…Mutations in any one of three conserved tryptophan residues in the prepro-domain of cathepsin L-like lysosomal cysteine peptidases destabilizes the alpha-helical motif resulting in misfolding and elimination from the ER via ERAD and the UPS [14]. To determine if the C. elegans cathepsin L-like protease, CPL-1, could be mutated in a similar fashion, we aligned the first 60 amino acids of the pro-domain of cpl-1 with those from the human cathepsin L-like cysteine proteases, cathepsins K, L, S and V (Figure 1A).…”

“…In mammalian systems, mutations in the prepro-region of lysosomal papain-like cysteine peptidases induce protein misfolding and convert them to luminal ERAD substrates that are efficiently degraded by the ubiquitin-proteasome system (UPS) [14]. In C. elegans , the best-described lysosomal cysteine peptidase is the cathepsin L-like protease, CPL-1 [15], [16].…”

Cathepsin S Supports Acid-independent Infection by Some Reoviruses

“…NIH3T3 and 293 cells were maintained in Dulbecco's minimal essential medium (Invitrogen) supplemented to contain 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin. 293 cells stably expressing Cat S (293:CatS) were a gift from Dr. Bernd Wiederanders (62) and were maintained in the same manner as the parental 293 cells except that 500 g/ml G418 (Calbiochem) was included in the medium.…”

Specialized roles for cysteine cathepsins in health and disease

“…In addition to its role in autoinhibition, the N-terminal proregion of cathepsin L appears to be necessary for the correct folding of the protein (35), a finding that is also true for papain (36). Mutations destabilizing the interface between the helices prevent correct folding of the protein (37,38). Folding of the proregion may precede folding of the full protein, thereby providing a scaffold that then directs the folding of the remaining domains.…”