Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
U9-ORF is a novel ligand for integrin αvβ3, which is upregulated on endothelial cells during inflammation, and thus could mediate protective effects of U90926 in sepsis, and thus could mediate protective effects of U90926 in sepsis.
Abstract
Gene expression in macrophages is a highly dynamic and tightly regulated process to prevent inflammation-driven pathology, such as septic shock. Previous studies in our lab identified the putative long non-coding RNA (lncRNA) gene U90926 as highly induced in macrophages following pattern recognition receptor activation by lipopolysaccharide (LPS) and other microbial ligands. Using an LPS-induced sepsis model, we found that U90926 knockout mice exhibited greater disease severity and mortality, indicating a protective role of the gene. Surprisingly, analysis of U90929 sequence identified an 87 amino acid open reading frame (ORF), encoding a novel protein, U9-ORF, containing a functional N-terminal secretion signal. We therefore hypothesized that protective effects of U90926 in sepsis are mediated by the secretion of U9-ORF and binding to receptors on other cells.
To screen for potential receptors for U9-ORF, we developed recombinant U9-ORF-Fc fusion proteins to measure binding via flow cytometry, and for use in vitro functional assays. Transfection of HEK293 cells was used to express potential receptors for U9-ORF, and cellular adhesion assays were used as a functional readout.
Multiple sequence alignments of predicted U9-ORF homologs revealed a conserved C-terminal RGD motif, suggesting that U9-ORF could bind specific integrins. Flow cytometry assays demonstrated that U9-ORF preferentially bound cells expressing integrin αvβ3, but not αIIbβ3, in an RGD- and divalent cation-dependent manner. Plate-bound U9-ORF-Fc supported cellular adhesion and spreading of cells expressing αvβ3 integrin, in an RGD-dependent manner.
U9-ORF is a novel ligand for integrin αvβ3, which is upregulated on endothelial cells during inflammation, and thus could mediate protective effects of U90926 in sepsis. Ongoing experiments aim to determine the effects of U9-ORF on endothelial barrier integrity in the presence of inflammatory stimuli.
NIAID/NIH R03 AI190196-01; NIAID/NIH R21 AI151116-01; Department of Education CMB-GAANN Fellowship
Cellular Adhesion, Migration, and Inflammation (CAM)
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infection in humans, but under conditions of immune suppression, it may reactivate and contribute to severe diseases, including Kaposi's sarcoma (KS) and B-cell malignancies. The KSHV genome encodes a single G protein-coupled receptor (GPCR), open reading frame 74 (ORF74), which shows homology to human chemokine receptors. Since its identification in 1996, ORF74 has subsequently been shown to interact with a broad range of human CXC chemokines, as well as CCL1 and the viral chemokine vCCL2. Compared with many human chemokine receptors, ORF74 displays high basal activity. These properties allow ORF74 to deregulate host cellular pathways through constitutive and chemokine-modulated signaling. In this study, we evaluated several human chemokines that, to our knowledge, had not previously been tested in ORF74-dependent cellular assays. Whereas CXCL9, CXCL14, CXCL16 and CXCL17 did not interact with ORF74, CXCL13 was identified as an additional ORF74 agonist and CXCL11 as an inverse agonist. CXCL13 dose-dependently induced ORF74-mediated Ca2+ release, β-arrestin1/2 recruitment and chemotaxis, and enhanced basal nuclear factor κB (NF-κB) activity in ORF74-expressing cells. In contrast, CXCL11 showed no detectable ORF74 agonist activity in the calcium mobilization or chemotaxis assay, but antagonized CXCL1-induced responses in both readouts. CXCL11 also elicited inverse agonist-like responses in β-arrestin1/2 recruitment assays and reduced basal NF-κB signaling. Our study thus reveals CXCL13 and CXCL11 as two additional chemokine ligands for ORF74, further expanding the pharmacological profile of this viral GPCR.
Qianqian Kong, Brent Van Bosstraeten, S. de Jonghe et al.· Biochemical Pharmacology· 0 citations
In vitro studies show that SLC15A4 inhibition results in the intracellular degradation of the TASL adapter protein and blockade of inflammatory cytokine production in response to TLR7/8/9 agonists, and these results highlight the early progress developing novel, drug-like inhibitors of SLC15A4.
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Extracellular protein expression in Escherichia coli is an elegant solution that addresses the complex issue of protein misfolding while simultaneously simplifying downstream processing steps. Human TNF-α was chosen as the target protein for export since it is a therapeutically important cytokine. Different genomic knockouts were tested for the ability to sustain and enhance protein expression, and BW25113 Δ(elaA + cysW) knockout was found to give a sustained and high level of expression. To improve secretion, various tags were tested, and the MBP tag at the N-terminal end was found to give maximum enhancement in the export of hTNF-α. Even the linker peptide was found to play a critical role in export, with the Ek linker giving the highest extracellular secretion, while the intein sequence completely blocked export. The co-expression of pSecAB, which is involved in protein transport to the periplasm, was also found to be helpful in enhancing extracellular protein titers. Interestingly, pelB performed poorly as compared to the native signal sequence of MBP, which gave better results. Culture conditions were optimized, and it was observed that growing cells in TB medium at a temperature of 25 °C, coupled with a pulse of concentrated nutrients at 24 h, led to a very high extracellular accumulation of ∼1.3 g/L of MBP-hTNF-α in shake flask culture. The protein was purified and tested using L929 cells for bioactivity. Thus, a combination of genomic and bioprocess strategies allowed us to obtain high levels of soluble and active extracellular expression of hTNF-α, making this a very attractive strategy for protein production.
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