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We performed a computational analysis on the first 70 aa at the N-t (N-terminal analysis) of the whole proteome and on 70 aa windows starting from all internal TMDs (Internal TMDs analysis) to identify potential SPC cleavage sites not associated with signal peptides (cryptic cleavage sites) by using a pre-existing a signal peptide prediction software SignalP and comparing the output of two networks (no-TM and TM modes). Our hits represent membrane protein with type-II oriented TMD predicted to be cleaved in no-TM network mode but not in TM network mode. Moreover, we rank the hits based on the number of disease-linked mutations.

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We used the command-line version of the SignalP4.1 program. The program takes a proteins sequence (FASTA formatted) of 70 amino acids length as input and predicts the cleavage site and reports the Y-score (combined cleavage site score) in its output. To extract proteins with cryptic cleavage sites, we created two types of peptide sequences by considering the first 70 amino acids of all:

• proteins that lack a canonical signal peptide (N-terminal analysis)
• transmembrane domains of proteins that lack a canonical signal peptide (Internal TMDs analysis)

We predicted the Y-scores of both types of peptide sequences using the SignalP-TM (TM network mode) and SignalP-noTM (no-TM network mode) version (see Methods for details). A protein/transmembrane domain was considered to have a non-canonical cleavage site if its corresponding peptide’s Y-score in the SignalP-TM output was less than 0.6 and in SignalP-noTM output was more than 0.5. We retrieved all the proteins and their associated feature information from UniProt/Swiss-Prot. We assigned mutational information provided in (a) UniProt, COSMIC, and ClinVAR databases.

Validation of the hits (proteins predicted to have non-canonical cleavage sites) were then performed by ectopically expressing the WT (Wild Type) and mutant constructs in WT HEK293T cells or lacking the regulatory SPC subunit SPCS1. Cell lysates were then analyzed via Western blot to detect possible SPCS1-dependent cleavage fragments (see Methods for details).

Note: The information about the data downloaded from UniProt, COSMIC and ClinVar can be retrieved from Materials and Methods of the accompanying manuscript.

Marius Lemberg: [email protected] (Lemberg lab, Heidelberg/Cologne)

Matthias Feige: [email protected] (CPB lab, Munich)

Gurdeep Singh: [email protected] (Russell lab, Heidelberg)

Zanotti A, Coelho JPL, Kaylani D, Singh G, Tauber M, Hitzenberger M, Avci D, Zacharias M, Russell RB, Lemberg MK, Feige MJ The human signal peptidase complex acts as a quality control enzyme for membrane proteins. Science (2022). doi: 10.1126/science.abo5672 PMID: 36454823