This communication reports on a possible distinct role of HMGB1 protein. Biophysical studies revealed that HMGB1 binds and stabilizes the G-quadruplex of the KRAS promoter element that is responsible for most of the transcriptional activity. Biological data showed that inhibition of HMGB1 increases KRAS expression. These results suggest that HMGB1 could play a role in the gene transcriptional regulation via the functional recognition of the G-quadruplex.

ABSTRACT:Cytochromes P450 (P450s) contribute to the metabolic activation and inactivation of various endogenous substrates. Despite years of research, the physiological role of CYP2S1 remains unknown. CYP2S1 has demonstrated NADPH P450-reductase-independent metabolism of cyclooxygenase (COX)-derived prostaglandins [e.g., prostaglandin G 2 (PGG 2 )] at nanomolar concentrations. Arachidonic acid is converted to prostaglandin precursors [PGG 2 and prostaglandin H 2 (PGH 2 )] through COX. These precursors are used to synthesize numerous prostanoids, including PGE 2 . Prostaglandin E 2 (PGE 2 ) promotes cell proliferation and cell migration and inhibits apoptosis. CYP2S1 metabolism of PGG 2 presumably sequesters PGG 2 and PGH 2 , making them unavailable for synthesis of prostanoids such as PGE 2 . Whether CYP2S1 contributes to prostaglandin metabolism and influences cell physiological remains to be determined. The purpose of this study was to evaluate the physiological role of CYP2S1, if any, in human bronchial epithelial cells [SV40-derived bronchial epithelial cell line (BEAS-2B)]. To do this, we used small interfering RNA to deplete CYP2S1 mRNA and protein by approximately 75% and evaluated the impact of CYP2S1 depletion on cell proliferation and migration. CYP2S1 depletion enhanced both cell proliferation and migration in BEAS-2B cells. Consistent with the proposed role of CYP2S1 in PGE 2 synthesis, the reduction in CYP2S1 expression doubled intracellular PGE 2 levels. Pharmacological administration of PGE 2 enhanced cell proliferation in BEAS-2B cells but failed to promote migration. Our data reveal an important role for CYP2S1 in the regulation of cell proliferation and migration, occurring in part through modulation of prostaglandin synthesis.

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20–30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin-a, an actin-crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA-PH domains of Grb7, and the immunoglobulin-like repeat 16–19 domains of Flna. We demonstrate that full-length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co-localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling.

Summary 24The liver x receptors (LXRs) are key regulators of systemic lipid metabolism. We identified 25 transmembrane protein 135 (TMEM135), a peroxisomal protein with an unknown function, as a 26 novel LXR target gene. The LXRs directly induce TMEM135 transcription in humans via an LXR 27 response element in the 5' untranslated region, but do not increase Tmem135 in murine cells. 28Functionally, knockdown of TMEM135 in vitro and in vivo results in many features typical of 29 peroxisomal disorders such as steatosis and reduced peroxisomal β-oxidation. Mechanistically, 30 proteomic and Western blot analyses indicated that TMEM135 mediates import of peroxisome 31 matrix proteins necessary for β-oxidation and bile acid synthesis. These findings indicate that 32 TMEM135 is an LXR-inducible regulator of peroxisome catabolic and anabolic processes by 33 mediating an auxiliary matrix protein import pathway, and thus may represent a novel therapeutic 34 target for disorders associated with peroxisome dysfunction. 35 Introduction 36The liver x receptors (LXR) α (NR1H3) and β (NR1H2) belong to the nuclear receptor 37 superfamily and are key regulators of cholesterol and fatty acid metabolism (1). Natural LXR 38 ligands include oxysterols (2) that increase during cholesterol loading and activate LXR-mediated 39 transcription. The LXRs heterodimerize with the retinoic acid receptor (RXR) and bind to LXR 40 response elements (LXRE) within the promoters of target genes (2). The LXREs belong to the 41 direct repeat 4 (DR4) class that consist of two hexanucleotide half-sites separated by a 4-42 nucleotide spacer (3,4). The protein products of known LXR target genes affect systemic lipid 43 metabolism. Well known examples include increasing cholesterol efflux via ATP-binding cassette 44 (ABC) sub-family A, member 1 (ABCA1) (5), and sub-family G, member 1 (ABCG1) (6); limiting 45 cholesterol uptake via induction of myosin regulatory light chain interacting protein (MYLIP) that 46 targets the low-density lipoprotein receptor (LDLR) for ubiquitin-mediated degradation (7); and 47 increased lipogenesis via induction of sterol regulatory element binding protein 1c (SREBP1c, 48 encoded by sterol regulatory element binding transcription factor 1 gene or SREBF1) (8,9). Also, 49. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/334979 doi: bioRxiv preprint first posted online May. 30, 2018; 3 the NR1H3 isoform of the LXRs is itself an LXR target gene (10,11), leading to auto-amplification 50 of LXR actions in certain species and tissues. Although many LXR target genes have been 51 discovered, additional target genes may remain unidentified. 52We have previously investigated LXR actions within the steroidogenic corpus luteum of 53 the ovary (12)(13)(14). In an attempt to uncover novel LXR target genes, rhesus macaque luteal cells ...

Background: Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3'UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells. Results: RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells. Conclusions: These data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.