Anti-Inflammatory Extract from Soil Algae Chromochloris zofingiensis Targeting TNFR/NF-κB Signaling at Different Levels.

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 IKKß 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.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

Screening for MAPK modulators using an in-cell western assay.

Several essential cellular functions are critically influenced by the Ras-Raf-MEK-ERK cascade, and pathological problems have been implicated with loss as well as gain of its activity. Therefore, there is a great demand for chemical probes that act as modulators of the cascade in order to correct medical problems associated with its malfunctions. Here we describe the use of the In-Cell Western assay, a fast and easy method for the detection of ERK activity. This method is useful for the screening of chemical libraries for compounds that modulate the intensity and duration of growth factor-induced MAPK activity. Examples are provided from a screen for MAPK modulators in Vichem Chemie Research´s Nested Chemical Library™.

TIS7 interacts with the mammalian SIN3 histone deacetylase complex in epithelial cells.

The mammalian SIN3 complex consists of histone deacetylases (HDAC1, HDAC2), several known proteins (SAP30, N-CoR) and as yet unidentified proteins. Here we show that the mouse tetradecanoyl phorbol acetate induced sequence 7 (TIS7) protein is a novel transcriptional co-repressor that can associate with the SIN3 complex. We have identified tis7 as a gene that is up-regulated upon loss of polarity in a mouse mammary gland epithelial cell line expressing an estrogen-inducible c-JunER fusion protein. In unpolarized cells, TIS7 protein levels increase and TIS7 translocates into the nucleus. Overexpression of tis7 causes loss of polarity and represses a set of genes, as revealed by cDNA microarray analysis. We have shown that TIS7 protein interacts with several proteins of the SIN3 complex (mSin3B, HDAC1, N-CoR and SAP30) by yeast two-hybrid screening and co-immunoprecipitations. TIS7 co-immunoprecipitated HDAC complex is enzymatically active and represses a GAL4-dependent reporter transcription. The transcriptional repression of endogenous genes by tis7 overexpression is HDAC dependent. Thus, we propose TIS7 as a transcriptional co-repressor affecting the expression of specific genes in a HDAC activity-dependent manner during cell fate decisions, e.g. scattering.