Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1 alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly

The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.

<p>Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.10⁸ yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.

<p>Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,<i>ahc1</i>Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.10⁸ yeast cells in average was used, except TAP/Spt8,<i>gcn5</i>Δ, TAP/Spt8,<i>ahc1</i>Δ, TAP/Spt8,<i>spt7</i>Δ and TAP/Gcn5,<i>ahc1</i>Δ strains, where 1.3 mL of cell lysates from 9.10⁸ yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Subcellular localization of pre-IL-1α and IL-1αMat in <i>Saccharomyces cerevisiae.</i>

<p>The IL-1α precursor (pre-IL-1α) is exclusively localized in the nucleus of yeast cells, which is in contrast to the observed cytoplasmic localization of mature IL-1α (IL-1αMat). Control cells (ctrl) carry the empty pUG36 vector. The cell nuclei are stained with DAPI.</p