Significance: The transcription factor nuclear factor kappa B (NF-κB) plays a vital role in cellular immune, stress, and proliferative responses by regulating genes. To maintain correct execution of the genetic program and to prevent pathologies (e.g., chronic inflammation), the activation of NF-κB has to be transient and precisely controlled. We describe a unique inhibitory mechanism that mediates NF-κB–dependent regulation of inflammation-associated genes and uses arginine methylation of the RelA subunit of NF-κB by protein arginine methyltransferase 1. This modification reduces the DNA-binding affinity of RelA, altering the recruitment to gene promoters and the transcriptional potency of NF-κB. This may be developed into an approach to inhibit the pathological functions of NF-κB.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45A^KO Caco-2 cells. In keeping with impaired myosin VB function, UNC45A^KO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45A^KO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, are increasing in populations worldwide. The treatment of patients with AD and other forms of skin inflammation is mainly based on the use of topical corticosteroids or calcineurin inhibitors, which can cause significant side effects with long-term use. Therefore, there is a great need for the development of more effective and less toxic anti-inflammatory agents suitable for the treatment of chronic skin lesions. Here, we screened a number of strains from the ASIB 505 terrestrial algae collection and identified a green algae Chromochloris zofingiensis with pronounced anti-inflammatory properties. We found that a crude nonpolar extract of C. zofingiensis (ID name NAE_2022C), grown upon nitrogen deprivation, acts as a bioactive substance by inhibiting TNFR/NF-κB responses in human skin keratinocyte HaCaT cells. We also found that NAE_2022C suppressed the secretion of pro-inflammatory cytokine tumor necrosis factor α (TNFα) and several Th1- and Th2-related chemokines in a reconstituted human epidermis. The TNFR/NF-κB pathway analysis showed multiple inhibitory effects at different levels and disclosed a direct targeting of IKKβ by the extract. Bioassay-guided fractionation followed by high-resolution mass spectrometry detected diacylglyceryl-trimethylhomoserine (DGTS), Lyso-DGTS (LDGTS), 5-phenylvaleric acid, theophylline and oleamide as leading metabolites in the active fraction of NAE_2022C. Further analysis identified betaine lipid DGTS (32:0) as one of the active compounds responsible for the NAE_2022C-mediated NF-κB suppression. Overall, this study presents an approach for the isolation, screening, and identification of anti-inflammatory secondary metabolites produced by soil algae.

The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (K_D) of 2.228 × 10^–6 M and 0.794 × 10^–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC)verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Congenital glucose-galactose malabsorption is a rare autosomal recessive disorder caused by mutations in SLC5A1 encoding the apical sodium/glucose cotransporter SGLT1. We present clinical and molecular data from eleven affected individuals with congenital glucose-galactose malabsorption from four unrelated, consanguineous Turkish families. Early recognition and timely management by eliminating glucose and galactose from the diet are fundamental for affected individuals to survive and develop normally. We identified novel SLC5A1 missense variants, p.Gly43Arg and p.Ala92Val, which were linked to disease in two families. Stable expression in CaCo-2 cells showed that the p.Ala92Val variant did not reach the plasma membrane, but was retained in the endoplasmic reticulum. The p.Gly43Arg variant, however, displayed processing and plasma membrane localization comparable to wild-type SGLT1. Glycine-43 displays nearly invariant conservation in the relevant structural family of cotransporters and exchangers, and localizes to SGLT1 transmembrane domain TM0. p.Gly43Arg represents the first disease-associated variant in TM0; however, the role of TM0 in the SGLT1 function has not been established. In summary, we are expanding the mutational spectrum of this rare disorder.

Urinary tract infections are among the most frequently occurring forms of infection, and inflammation and tissue damage contribute significantly to symptoms, e.g., dysuria and urge. Canephron N is an orally bioavailable herbal medicine with anti-inflammatory, spasmolytic, anti-adhesive, and anti-nociceptive therapeutic effects that is approved for the treatment of uncomplicated urinary tract infections. Here, we used renal tubular epithelial HK-2 cells to study the anti-inflammatory and cytoprotective effects and molecular mechanisms of its active component, BNO 2103. BNO 2103 suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation by lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) and prevented inhibitory κB kinase (IKK)-dependent phosphorylation and degradation of inhibitor of nuclear factor kappa B alpha (IκBα). BNO 2103 also suppressed the inflammation-specific S536 phosphorylation of the NF-κB subunit p65 and the production of a specific set of inflammatory cytokines. Unlike other NF-κB inhibitors, BNO 2103 demonstrated cytoprotection against TNFα-induced cytotoxicity. Our data suggest that BNO 2103 acts primarily through the mitogen-activated protein kinase p38 (p38 MAPK)-MAPK-activated protein kinase 2 (MK2) axis by promoting receptor-interacting serine/threonine protein kinase 1 (RIPK1) phosphorylation at S320. Simultaneously, it suppresses S166 autophosphorylation and subsequent activation of RIPK1, which is required for apoptotic and necroptotic responses to TNFα. This study confirms Canephron N as an effective alternative to traditional anti-inflammatory drugs and provides initial evidence of its ability to inhibit apoptosis and necroptosis in the urogenital system. It also presents a detailed pathway investigation that identifies the specific targets of Canephron N within the NF-κB signaling cascade.