Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid–Liquid Phase Separation

Abstract

Intrinsically disordered proteins (IDPs) move through an ensemble of conformations which allows multitudinous roles within a cell. Keratinocytes, the predominant cell type in mammalian epidermis, have had only a few individual proteins assessed for intrinsic disorder and its possible contribution to liquid–liquid phase separation (LLPS), especially in regard to what functions or structures these proteins provide. We took a holistic approach to keratinocyte IDPs starting with enrichment via the isolation of thermostable proteins. The keratinocyte protein involucrin, known for its resistance to heat denaturation, served as a marker. It and other thermostable proteins were identified by liquid chromatography tandem mass spectrometry and subjected to extensive bioinformatic analysis covering gene ontology, intrinsic disorder, and potential for LLPS. Numerous proteins unique to keratinocytes and other proteins with shared expression in multiple cell types were identified to have IDP traits (e.g., compositional bias, nucleic acid binding, and repeat motifs). Among keratinocyte-specific proteins, many that co-assemble with involucrin into the cell-specific structure known as the cornified envelope scored highly for intrinsic disorder and potential for LLPS. This suggests intrinsic disorder and LLPS are previously unrecognized traits for assembly of the cornified envelope, echoing the contribution of intrinsic disorder and LLPS to more widely encountered features such as stress granules and PML bodies.

Discovery of natural bispecific antibodies: Is psoriasis induced by a toxigenic Corynebacterium simulans and maintained by CIDAMPs as autoantigens?

“…Antigen‐bound psoriasis‐IgG was detected after treatment of the membrane with goat‐anti‐human IgG, peroxidase‐coupled, and development with Roche LumiLight as described 38 . Note the presence of a WB‐band only when the samples were heated or sonicated and immediately used, conditions known to dissociate complexes of Intrinsically Disordered Proteins and Regions (IDP(R)s) 37 …”

“…HRNR‐peptides bind to ribosomal proteins, a likely mode of action of bactericidal activity of cationic intrinsically disordered antimicrobial peptides, (CIDAMPs) 30 . IDPs can form hundreds of different protein complexes with other IDPs, which are rather stable at physiological conditions but can be dissociated upon heat treatment, 36,37 conditions necessary for antibody‐binding to C. simulans antigens as shown in Figure 1, or to see CIDAMP‐binding to ribosomal proteins 38 . So, I would propose that in the quaternary structure of a multimeric assembly complex of two IDP(R)s short, exposed peptide chains of each IDP form discontinuous epitopes from chimeric antigens, which both together will lead to the formation of BsAbs.…”

“…Thus, it is tempting to speculate that a CIDAMP‐based, persisting epithelial defence reaction within the stratum corneum and stratum granulosum towards virulent C. simulans could eventually result in the generation of CIDAMP‐like autoantigens. Since CIDAMPs are generated from cationic IDP(R)s, which are highly abundant in keratinocytes, 37 and are produced within the fully differentiated epidermal layers, it would be plausible to see CIDAMP‐directed immune reactions, such as IgG‐deposits, within the stratum corneum 3,66 and autoreactive T‐cell‐mediated 67,68 responses within the stratum granulosum. Keratinocyte‐derived CIDAMPS as autoantigens would also help to explain the typical gyrate, demarcated lesion morphology of annular psoriasis, 69 where I would propose that the centre is missing CIDAMP sources due to a loss of the stratum granulosum.…”

Discovery of Natural Bispecific Antibodies: Is Psoriasis Induced by a ToxigenicCorynebacterium simulansand Maintained by CIDAMPs as Autoantigens?

“…HRNR-peptides bind to ribosomal proteins, a likely mode of action of bactericidal activity of Cationic Intrinsically Disordered Antimicrobial Peptides, CIDAMPs 60 . IDPs can form hundreds of different protein complexes with other IDPs, which are rather stable at physiological conditions but can be dissociated upon heat treatment 59,52 , conditions necessary for antibody-binding to C. simulans antigens as shown in Fig.1, or to see CIDAMP-binding to ribosomal proteins 50 . So, I would propose that in the quaternary structure of a multimeric assembly complex of two IDP(R)s short, exposed peptide chains of each IDP form discontinuous epitopes from chimeric antigens, which both together will lead to the formation of BsAbs.…”

“…Thus, it is tempting to speculate that a CIDAMP-based, persisting epithelial defense reaction within the stratum corneum and stratum granulosum towards virulent C. simulans could eventually result in the generation of CIDAMP-like autoantigens. Since CIDAMPs are generated from cationic IDP(R)s, which are highly abundant in keratinocytes 52 , and are produced within the fully differentiated epidermal layers, it would be plausible to see CIDAMP-directed immune reactions, such as IgG- deposits, within the stratum corneum 3,88 and autoreactive T-cell-mediated 89,90 responses within the stratum granulosum. Keratinocyte-derived CIDAMPS as autoantigens would also help to explain the typical gyrate, demarcated lesion morphology of annular psoriasis 91 , where I would propose that the center is missing CIDAMP sources due to a loss of the stratum granulosum.…”