Critical Analysis of Cytoplasmic Progression of Inflammatory Signaling Suggests Potential Pharmacologic Targets for Wound Healing and Fibrotic Disorders.

Successful skin wound healing is dependent on an interplay between epidermal keratinocytes and dermal fibroblasts as they react to local extracellular factors (DAMPs, PAMPs, cytokines, etc.) surveyed from that environment by numerous membrane receptors (e.g.

Investigating Protein-Protein Interactions of Autophagy-Involved TNIP1.

Myriad proteins are involved in the process of autophagy, which they participate in via their protein-protein interactions (PPI). Herein we outline a methodology for examining such interactions utilizing the case of intrinsically disordered protein (IDP) TNIP1 and its interaction with linear M1-linked polyubiquitin. This includes methods for recombinant production, purification, immuno-identification, and analysis of an IDP associated with autophagy, its ordered binding partner, and means of quantitatively analyzing their interaction.

Repressive Control of Keratinocyte Cytoplasmic Inflammatory Signaling.

The overactivity of keratinocyte cytoplasmic signaling contributes to several cutaneous inflammatory and immune pathologies. An important emerging complement to proteins responsible for this overactivity is signal repression brought about by several proteins and protein complexes with the native role of limiting inflammation. The signaling repression by these proteins distinguishes them from transmembrane receptors, kinases, and inflammasomes, which drive inflammation.

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.

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.

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.

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.

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.

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.

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.

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.

Interleukin-17 signaling triggers degradation of the constitutive NF-κB inhibitor ABIN-1.

IL-17 activates NF-κB and inducing expression of proinflammatory genes. IL-17 drives disease in autoimmune conditions, and anti-IL-17 antibodies have shown impressive success in the clinic. Although produced by lymphocytes, IL-17 predominantly signals in fibroblasts and epithelial cells.

TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors.

TNFα-induced protein 3-interacting protein 1 (TNIP1) represses signaling pathways initiated by specific nuclear and transmembrane receptors. This effect results in reduced activity of distinct transcription factors such as retinoic acid receptors (RAR), peroxisome-proliferator-activated receptors (PPAR), and NFκB. TNIP1-null and TNIP1-knockin defective for ubiquitin-binding mice show increased liver apoptosis, and enlarged spleen and lymph nodes, respectively.

Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions.

Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B') is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced.

Transgene delivery to cultured keratinocytes via replication-deficient adenovirus vectors.

Transient transgene expression can facilitate investigation of that gene-product function or effect on keratinocyte biology. Several chemical and biologic delivery systems are available, and among them adenoviruses offer particular advantages in efficiency and transgene capacity. Here we describe the advantages of bicistronic adenovirus and inclusion of the polycation hexadimethrine bromide to aid in the detection of positively transduced cells and enhance transduction efficiency.

Sp sites contribute to basal and inducible expression of the human TNIP1 (TNFα-inducible protein 3-interacting protein 1) promoter.

TNIP1 [TNFα (tumour necrosis factor α)-induced protein 3-interacting protein 1] is a co-repressor of RAR (retinoic acid receptor) and PPAR (peroxisome-proliferator-activated receptor). Additionally, it can reduce signalling stemming from cell membrane receptors such as those for TNFα and EGF (epidermal growth factor). Consequently, it influences a variety of receptor-mediated events as diverse as transcription, programmed cell death and cell cycling.

Human TNFα-induced protein 3-interacting protein 1 (TNIP1) promoter activation is regulated by retinoic acid receptors.

Coregulator proteins play key roles in transcriptional control by members of the nuclear receptor superfamily. Previously, we demonstrated that tumor necrosis factor α (TNFα)-induced protein 3-interacting protein 1 (TNIP1) is a corepressor of agonist-bound retinoic acid receptors (RARs). Additionally, TNIP1 has been shown to repress peroxisome proliferator-activated receptors (PPAR) and NF-κB activity and interact with HIV proteins nef and matrix.

Organotypic modeling of human keratinocyte response to peroxisome proliferators.

Peroxisome proliferators (PPs) are a diverse chemical group including hypolipidemic drugs and some fatty acids. Their stimulation of PP-activated receptors (PPARs) and subsequent control of gene expression regulates metabolism and differentiation in many cells. PPs have multiple opportunities to target human epidermal keratinocytes because of delivery through dietary, clinical, and/or topical exposure routes.

Emerging roles for TNIP1 in regulating post-receptor signaling.

A vast number of cellular processes and signaling pathways are regulated by various receptors, ranging from transmembrane to nuclear receptors. These receptor-mediated processes are modulated by a diverse set of regulatory proteins. TNFα-induced protein 3-interacting protein 1 is such a protein that inhibits both transduction by transmembrane receptors, such as TNFα-receptor, EGF-R, and TLR, and nuclear receptors' PPAR and RAR activity.

TNIP1, a retinoic acid receptor corepressor and A20-binding inhibitor of NF-κB, distributes to both nuclear and cytoplasmic locations.

An increasingly wide range of functions, from repression of NF-κB signaling to protection from apoptosis, is being recognized for tumor necrosis factor α-induced protein 3-interacting protein 1 (TNIP1). The authors recently demonstrated TNIP1 interaction with and repression of liganded retinoic acid receptors, distinguishing it from the more typical NCoR and SMRT corepressors, which function only in the absence of ligand. To improve their understanding of TNIP1's roles in physiologic and pathologic events, the authors examined its distribution in normal and malignant human tissues and cultured cells.

PPARγ and NF-κB regulate the gene promoter activity of their shared repressor, TNIP1.

Human TNFAIP3 interacting protein 1 (TNIP1) has diverse functions including support of HIV replication through its interaction with viral Nef and matrix proteins, reduction of TNFα-induced signaling through its interaction with NF-κB pathway proteins, and corepression of agonist-bound retinoic acid receptors and peroxisome proliferator-activated receptors (PPAR). The wide tissue distribution of TNIP1 provides the opportunity to influence numerous cellular responses in these roles and defining control of TNIP1 expression would be central to improved understanding of its impact on cell function. We cloned 6kb of the human TNIP1 promoter and performed predictive and functional analyses to identify regulatory elements.

TNIP1 is a corepressor of agonist-bound PPARs.

Nuclear receptor (NR) coregulators include coactivators, contributing to holoreceptor transcriptional activity, and corepressors, mediating NR target gene silencing in the absence of hormone. We identified an atypical NR coregulator, TNFα-induced protein 3-interacting protein 1 (TNIP1), from a peroxisome proliferator activated receptor (PPAR) α screen of a human keratinocyte cDNA library. TNIP1's complex nomenclature parallels its additional function as an NF-κB inhibitor.

Scanning for transcription factor binding by a variant EMSA.

Detection of in vitro protein-DNA interaction is one of many investigational analyses for transcription factor regulation of gene promoters. The electrophoretic mobility shift assay (EMSA) has proven widely popular in this respect by integrating individual techniques (protein isolation, nucleic acid radiolabeling, and gel electrophoresis) into one protocol. However, relatively short DNA oligomers are often used which in many cases presupposes what one sequence out of a promoter of possibly thousands of base pairs is the candidate region interacting with a transcription factor.

Liganded RARalpha and RARgamma interact with but are repressed by TNIP1.

Nuclear receptor (NR) transcriptional activity is controlled by agonist binding and concomitant exchange of receptor-associating corepressor proteins for NR box-containing, receptor AF-2-targeting coactivator proteins. We report here that TNIP1 is an atypical NR coregulator. Requirements for TNIP1-RAR interaction-its NR boxes, ligand, and the receptor's AF-2 domain-are characteristic of coactivators.

Corepressors of agonist-bound nuclear receptors.

Nuclear receptors (NRs) rely on coregulator proteins to modulate transcription of target genes. NR coregulators can be broadly subdivided into coactivators which potentiate transcription and corepressors which silence gene expression. The prevailing view of coregulator action holds that in the absence of agonist the receptor interacts with a corepressor via the corepressor nuclear receptor (CoRNR, "corner") box motifs within the corepressor.