Publications

Angiotensin converting enzyme 2 (ACE2) works in the renin angiotensin aldosterone system to decrease circulating levels of angiotensin II by removing the C-terminal phenylalanine and converting it to angiotensin (1-7). In addition, ACE2 has received increased interest in research due to its role in COVID-19 pathogenesis, as the binding site and cell entry gate for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While ACE2 inhibitors have been primarily used as pharmacological tools to study the renin-angiotensin system, small molecule ACE2 enhancers (aka activators) are highly desired because of their hypothesized therapeutic potential. This study was designed to identify peptide-based enhancers of ACE2. First, binding of human recombinant ACE2 to all possible tripeptides composed of the 20 proteinogenic amino acids, was evaluated using a proprietary immunofluorescence-based peptide microarray. Binding of 6xHis-tagged ACE2 to the 8000 tripeptides immobilized on a microchip was evaluated at 10 µg/ml and 100 µg/ml concentrations of the peptidase using a DyLight680-conjugated anti-6xHis-tag antibody. Hemagglutinin (HA) immobilized on the microchip served as a positive control peptide in the microarray and it was tracked using a DyLight800-conjugated anti-HA antibody. The read-out was performed with an Innopsys InnoScan 710-IR Microarray Scanner at scanning gains of 50/10 (red/green). In the result of the microarray a number of tripeptides were identified as potential ACE2 binders. Among them, 22 tripeptides were selected to represent several the most pronounced binders as well as a number of structurally similar tripeptides that did not show appreciable binding to ACE2 to serve as negative control. The effect of the selected peptides (at 1, 10 and 100 µM) on activity of human recombinant ACE2 was tested in a continuous enzymatic assay using a fluorogenic substrate. Contrary to our expectation, none of the peptides affected the activity of ACE2 in a significant manner. These results suggest that the selected peptides do not alter activity of ACE2, but they do not exclude the possibility that some of the peptides may still bind to the peptidase. Our subsequent experiments will apply differential scanning fluorometry (DSF) to determine whether these peptides physically interact with recombinant ACE2.

Background: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). Methods: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. Results: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. Conclusions: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS.