High throughput methods to study protein-protein interactions during host-pathogen interactions

The ability of a pathogen to survive and cause an infection is often determined by specific interactions between the host and pathogen proteins. Such interactions can be both intra- and extracellular and may define the outcome of an infection. There are a range of innovative biochemical, biophysical and bioinformatic techniques currently available to identify protein-protein interactions (PPI) between the host and the pathogen. However, the complexity and the diversity of host-pathogen PPIs has led to the development of several high throughput (HT) techniques that enable the study of multiple interactions at once and/or screen multiple samples at the same time, in an unbiased manner. We review here the major HT laboratory-based technologies employed for host-bacterial interaction studies. [Abstract copyright: Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.

Elevated levels of plasma symmetric dimethylarginine and increased arginase activity as potential indicators of cardiovascular comorbidity in rheumatoid arthritis

Abstract Background Rheumatoid arthritis (RA) patients are at high risk of developing cardiovascular disease (CVD). In RA, chronic inflammation may lead to endothelial dysfunction, an early indicator of CVD, owing to diminished nitric oxide (NO) production. Because l-arginine is the sole precursor of NO, we hypothesized that levels of l-arginine metabolic products reflecting NO metabolism are altered in patients with RA. Methods Plasma samples from patients with RA (n = 119) and age- and sex-matched control subjects (n = 238) were used for this study. Using LC-MS/MS, we measured plasma levels of free l-arginine, l-ornithine, l-citrulline, l-N G-monomethyl arginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). We compared global arginine bioavailability ratio (GABR) (i.e., ratio of l-arginine to l-ornithine + l-citrulline) and arginine methylation index (ArgMI) (i.e., ADMA + SDMA/MMA) in patients with RA vs. control subjects. Plasma arginase activity was measured using a sensitive arginase assay kit. The relationship of l-arginine metabolites and arginase activity to CVD risk factors was evaluated using Pearson’s chi-square test. Results Compared with healthy control subjects, the RA cohort showed significantly lower levels of plasma l-arginine (46.11 ± 17.29 vs. 74.2 ± 22.53 μmol/L, p < 0.001) and GABR (0.36 ± 0.16 vs. 0.73 ± 0.24, p < 0.001), elevated levels of ADMA (0.76 ± 0.12 vs. 0.62 ± 0.12 μmol/L, p < 0.001), SDMA (0.54 ± 0.14 vs. 0.47 ± 0.13 μmol/L, p < 0.001), and ArgMI (6.51 ± 1.86 vs. 5.54 ± 1.51, p < 0.001). We found an approximately fourfold increase in arginase activity (33.8 ± 1.1 vs. 8.4 ± 0.8 U/L, p < 0.001), as well as elevated levels of arginase-mediated l-arginine catalytic product l-ornithine (108.64 ± 30.26 vs. 69.3 ± 20.71 μmol/L, p < 0.001), whereas a nitric oxide synthase (NOS) catalytic product, the l-citrulline level, was diminished in RA (30.32 ± 9.93 vs. 36.17 ± 11.64 μmol/L, p < 0.001). Patients with RA with existing CVD had higher arginase activity than patients with RA without CVD (p = 0.048). Conclusions Global l-arginine bioavailability was diminished, whereas plasma arginase activity, ADMA, and SDMA levels were elevated, in patients with RA compared with healthy control subjects. Plasma SDMA was associated with hypertension and hyperlipidemia in patients with RA. This dysregulated l-arginine metabolism may function as a potential indicator of CVD risk in patients with RA

TNF and IFN-γ mediated <i>CXCL11</i> induction requires PRMT5 catalyzed p65 dimethylation at Arg¹⁷⁴.

<p>siRNA targeting the 3´ UTR of p65 was cotransfected in EC with cDNA encoding wild type, Arg¹⁷⁴Ala, or Arg¹⁷⁴Lys p65. Knockdown of endogenous p65 and reconstitution with p65 constructs is shown by immunoblot and densitometric analysis <b>(A)</b>. EC reconstituted with wild type and p65 mutants were treated with the indicated pro-inflammatory agonists for 3 hours followed by RNA collection and real-time PCR analysis to determine the mRNA levels of <i>CXCL11</i> <b>(B)</b> and <i>CXCL10</i> <b>(C)</b>. In <b>(D)</b>, FLAG-p65 was immunoprecipitated from EC transfected with two different PRMT5 siRNAs (sequence 1, lane 4; sequence 2, lane 5) following stimulation with TNF and IFN-γ for 30 minutes. Immunoprecipitates were separated by PAGE and the gel was silver-stained. FLAG-p65 bands were excised for mass spectrometric analysis. <b>(E)</b> Mass spectrometric quantitation of the peak area of dimethylated Arg¹⁷⁴ present in the tryptic ¹⁷²PL<b>R</b>LPPVLSHPIFDNR¹⁸⁵ peptide relative to the unmodified peptide. The 28 in the peptide sequence refers to the +28 Da mass shift consistent with dimethylation (2 x 14 Da) of Arg¹⁷⁴.*, <i>p</i> < 0.05; ***, <i>p</i> < 0.005; error bars represent S.E (<i>n</i> = 3).</p

p65 Arg¹⁷⁴ methylation is present on the <i>CXCL11</i> promoter following costimulation.

<p><b>(A)</b> EC were depleted of endogenous p65 and reconstituted with wild type or Arg¹⁷⁴Lys mutant p65. Cells were stimulated with TNF plus IFN-γ for 30 minutes and lysed in RIPA buffer. Wild type and mutant p65 was immunoprecipitated using the FLAG epitope or IgG. Immunoprecipitates were immunoblotted for p65 and for the SDMA modification. <b>(B-C)</b> Cells were reconstituted with <i>N</i>-FLAG wild type, Arg³⁰Lys, Arg³⁵Lys, and Arg¹⁷⁴Lys p65 constructs, and simultaneously exposed to TNF and IFN-γ. ChIP was performed using anti-FLAG antibody. Sequential ChIP (Re-ChIP) was carried out with addition of normal rabbit serum or anti-SYM10 antibody. Following crosslink reversal, DNA was purified and promoter fragment content was quantitated using qRT-PCR relative to input.</p

PRMT5 knockdown reduces association of p65 with the <i>CXCL11</i> promoter.

<p>The location of major regulatory elements and primer binding sites used for the chromatin immunoprecipitation (ChIP) assay of the <i>CXCL11</i> promoter are illustrated in <b>(A)</b>. IFN-γ response elements include the gamma interferon activation site (GAS) and interferon-sensitive response element (ISRE). The major TNF responsive element is the NF-κB binding site. ChIP assays were performed to assess p65 binding with the proximal promoter encompassing the κB binding site(s) of <i>CXCL11</i> <b>(B)</b>, <i>CXCL10</i> <b>(C)</b>, and <i>CCL2</i> <b>(D)</b> in PRMT5-intact or -depleted EC stimulated with TNF plus IFN-γ for 30 minutes. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; error bars represent S.E. (<i>n</i> = 3).</p

PRMT5 promotes expression of <i>CXCL11</i> in EC costimulated with TNF and IFN-γ.

<p><b>(A)</b> PRMT5-specific siRNA depleted protein levels in endothelial cells (EC) by ~90% as measured by immunoblotting (left panel) and densitometry (right panel). Expression of the chemokines <i>CXCL11</i> <b>(B)</b>, <i>CXCL10</i> <b>(C)</b> and <i>CCL2</i> <b>(D)</b> were measured by real-time PCR of RNA isolated from EC transfected with the indicated siRNAs and subsequently stimulated with TNF, IFN-γ, or TNF plus IFN-γ for 3 hours. An immunoblot showing CXCL11 protein expression in PRMT5 or control siRNA-transfected cells after 3 h of stimulation is shown in <b>(E)</b>. *, <i>p</i> < 0.05; ***, <i>p</i> < 0.005; error bars represent S.E. (<i>n</i> = 3–4).</p