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

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.

Intrinsic disorder in integral membrane proteins.

The well-defined roles and specific protein-protein interactions of many integral membrane proteins (IMPs), such as those functioning as receptors for extracellular matrix proteins and soluble growth factors, easily align with considering IMP structure as a classical "lock-and-key" concept. Nevertheless, continued advances in understanding protein conformation, such as those which established the widespread existence of intrinsically disordered proteins (IDPs) and especially intrinsically disordered regions (IDRs) in otherwise three-dimensionally organized proteins, call for ongoing reevaluation of transmembrane proteins. Here, we present basic traits of IDPs and IDRs, and, for some select single-span IMPs, consider the potential functional advantages intrinsic disorder might provide and the possible conformational impact of disease-associated mutations. For transmembrane proteins in general, we highlight several investigational approaches, such as biophysical and computational methods, stressing the importance of integrating them to produce a more-complete mechanistic model of disorder-containing IMPs. These procedures, when synergized with in-cell assessments, will likely be key in translating in silico and in vitro results to improved understanding of IMP conformational flexibility in normal cell physiology as well as disease, and will help to extend their potential as therapeutic targets.

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder.

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid-liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

The Anti-Inflammatory Protein TNIP1 Is Intrinsically Disordered with Structural Flexibility Contributed by Its AHD1-UBAN Domain.

TNFAIP3 interacting protein 1 (TNIP1) interacts with numerous non-related cellular, viral, and bacterial proteins. TNIP1 is also linked with multiple chronic inflammatory disorders on the gene and protein levels, through numerous single-nucleotide polymorphisms and reduced protein amounts. Despite the importance of TNIP1 function, there is limited investigation as to how its conformation may impact its apparent multiple roles. Hub proteins like TNIP1 are often intrinsically disordered proteins. Our initial in silico assessments suggested TNIP1 is natively unstructured, featuring numerous potentials intrinsically disordered regions, including the ABIN homology domain 1-ubiquitin binding domain in ABIN proteins and NEMO (AHD1-UBAN) domain associated with its anti-inflammatory function. Using multiple biophysical approaches, we demonstrate the structural flexibility of full-length TNIP1 and the AHD1-UBAN domain. We present evidence the AHD1-UBAN domain exists primarily as a pre-molten globule with limited secondary structure in solution. Data presented here suggest the previously described coiled-coil conformation of the crystallized UBAN-only region may represent just one of possibly multiple states for the AHD1-UBAN domain in solution. These data also characterize the AHD1-UBAN domain in solution as mostly monomeric with potential to undergo oligomerization under specific environmental conditions (e.g., binding partner availability, pH-dependence). This proposed intrinsic disorder across TNIP1 and within the AHD1-UBAN region is likely to impact TNIP1 function and interaction with its multiple partners.

Enhanced Wound Healing- and Inflammasome-Associated Gene Expression in TNFAIP3-Interacting Protein 1- (TNIP1-) Deficient HaCaT Keratinocytes Parallels Reduced Reepithelialization.

TNIP1 protein is a widely expressed, cytoplasmic inhibitor of inflammatory signaling initiated by membrane receptors such as TLRs which recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Keratinocyte TNIP1 deficiency sensitizes cells to PAMPs and DAMPs promoting hyperresponsive expression and secretion of cytokine markers (e.g., IL-8 and IL-6) relevant to cases of chronic inflammation, like psoriasis, where TNIP1 deficiency has been reported. Here, we examined the impact of TNIP1 deficiency on gene expression and cellular responses (migration and viability) relevant to acute inflammation as typically occurs in wound healing. Using siRNA-mediated TNIP1 expression knockdown in cultured HaCaT keratinocytes, we investigated TNIP1 deficiency effects on signaling downstream of TLR3 agonism with low-concentration poly (I:C), a representative PAMP/DAMP. The combination of TNIP1 knockdown and PAMP/DAMP signaling disrupted expression of specific keratinocyte differentiation markers (e.g., transglutaminase 1 and involucrin). These same conditions promoted synergistically increased expression of wound-associated markers (e.g., S100A8, TGFß, and CCN2) suggesting potential benefit of increased inflammatory response from reduced TNIP1 protein. Unexpectedly, poly (I:C) challenge of TNIP1-deficient cells restricted reepithelialization and reduced cell viability. In these cells, there was not only increased expression for genes associated with inflammasome assembly (e.g., ASC, procaspase 1) but also for A20, a TNIP1 partner protein that represses cell-death signaling. Despite this possibly compensatory increase in A20 mRNA, there was a decrease in phospho-A20 protein, the form necessary for quenching inflammation. Hyperresponsiveness to poly (I:C) in TNIP1-deficient keratinocytes was in part mediated through p38 and JNK pathways. Taken together, we conclude that TNIP1 deficiency promotes enhanced expression of factors associated with promoting wound healing. However, the coupled, increased potential priming of the inflammasome and reduced compensatory activity of A20 has a net negative effect on overall cell recovery potential manifested by poor reepithelialization and viability. These findings suggest a previously unrecognized role for TNIP1 protein in limiting inflammation during successful progression through early wound healing stages.

In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Analysis.

Nuclear receptors (NR) regulate gene expression critical in keratinocyte replication and differentiation. In addition to a ligand-binding domain, NR like other transcription factor families have a DNA-binding domain that must attain a particular conformation for effective interaction with the three-dimensional structure in promoters of target genes for control of their expression. Such protein-DNA assemblies extend the classic "lock and key" idea typified by protein-protein interactions. However, it is becoming increasingly clear that multi-subdomain transcription factors like NR frequently range along the length of the protein from structured, ordered regions expected for interaction with a preset partner to more flexible, intrinsically disordered regions which are more available for diverse posttranslational modifications and/or interaction with differing partners. The extended amino terminus of NR (the A/B subdomain) is one such intrinsically disordered region. Here we provide a primer on in silico-based recognition of amino acid composition and order associated with such conformational flexibility along with adaptations of readily accessible laboratory techniques (e.g., considerations for recombinant expression, sensitivity to protease and proteasome digestion) to facilitate initial prediction and testing for intrinsic disorder in various proteins of interest to keratinocyte biologists, like NR and other transcription factors.

TNIP1 in Autoimmune Diseases: Regulation of Toll-like Receptor Signaling.

TNIP1 protein is increasingly being recognized as a key repressor of inflammatory signaling and a potential factor in multiple autoimmune diseases. In addition to earlier foundational reports of TNIP1 SNPs in human autoimmune diseases and TNIP1 protein-protein interaction with receptor regulating proteins, more recent studies have identified new potential interaction partners and signaling pathways likely modulated by TNIP1. Subdomains within the TNIP1 protein as well as how they interact with ubiquitin have not only been mapped but inflammatory cell- and tissue-specific consequences subsequent to their defective function are being recognized and related to human disease states such as lupus, scleroderma, and psoriasis. In this review, we emphasize receptor signaling complexes and regulation of cytoplasmic signaling steps downstream of TLR given their association with some of the same autoimmune diseases where TNIP1 has been implicated. TNIP1 dysfunction or deficiency may predispose healthy cells to the inflammatory response to otherwise innocuous TLR ligand exposure. The recognition of the anti-inflammatory roles of TNIP1 and improved integrated understanding of its physical and functional association with other signaling pathway proteins may position TNIP1 as a candidate target for the design and/or testing of next-generation anti-inflammatory therapeutics.

TNIP1 reduction sensitizes keratinocytes to post-receptor signalling following exposure to TLR agonists.

Cell level inflammatory signalling is a combination of initiation at cell membrane receptors and modulation by cytoplasmic regulatory proteins. For keratinocytes, the predominant cell type in the epidermis, this would include toll-like receptors (TLR) and cytoplasmic proteins that propagate or dampen post-receptor signalling. We previously reported that increased levels of tumor necrosis factor α induced protein 3-interacting protein 1 (TNIP1) in HaCaT keratinocytes leads to decreased expression of stress response and inflammation-associated genes. This finding suggested decreased TNIP1 levels, as seen in some cutaneous disease states, may produce the opposite effect, sensitizing cells to triggers of inflammatory signalling including those sensed by TLR. In this study of TNIP1-deficient HaCaT keratinocytes we examined intracellular signalling consequences especially those expected to produce gene expression changes downstream of TLR3 or TLR2/6 activation by Poly (I:C) or FSL-1, agonists modeling skin relevant pathogens. We found TNIP1-deficient keratinocytes are hyper-sensitive to TLR activation compared to control cells with a normal complement of TNIP1 and receiving the same agonist stimulation. TNIP1-deficient keratinocytes have increased levels of activated (phosphorylated) cytoplasmic mediators such as JNK and p38 and greater nuclear translocation of NF-κB and phospho-p38 when exposed to TLR ligands. This is consistent with significantly increased expression of several inflammatory cytokines and chemokines, such as IL-6 and IL-8. These results describe how decreased TNIP1 levels promote a hyper-sensitive state in HaCaT keratinocytes evidenced by increased activation of signalling molecules downstream of TLR agonists and increased expression of pro-inflammatory mediators. TNIP1 keratinocyte deficiency as reported for some skin diseases may predispose these cells to excessive inflammatory signalling upon exposure to viral or bacterial ligands for TLR.