Stevens, J. J. | Search bioRxiv

bioRxiv2026

Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

Nitric oxide (NO*) is an important signaling molecule in many biological processes, including immune response. During response to classical activation stimuli lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma}), macrophages generate NO* via inducible nitric oxide synthase (iNOS). To comprehensively define the effects of NO*, we applied a multi-omic strategy integrating proteomics and transcriptomics to profile murine macrophages across conditions with or without LPS/IFN{gamma}-activation, with or without iNOS expression or exogenous NO* donor treatment. The results revealed NO* has broad, yet selected and controlled, regulatory effects, playing a key role in coordinating the systematic remodeling during macrophage classical activation. Among the proteins that are most suppressed in a NO*-dependent manner, electron transport chain (ETC) is the most enriched. NO* drives complex-specific remodeling of ETC, causing selected downregulation of complex I, II, and IV, through a different combination of transcriptional and post-transcriptional mechanisms for each complex. Functionally, we found NO* is required, but not sufficient, for the strong suppression of cellular respiration upon macrophage activation. Among the most consistently upregulated proteins are many enzymes involved in redox defense. AKR1A1 was identified as a top hit. We found Akr1a1 induction requires both NO* and LPS/IFN{gamma} stimulation. The S-nitroso-CoA reductase activity of AKR1A1 mitigates NO*-driven inhibition of pyruvate dehydrogenase complex by limiting the inhibitory modifications targeting its lipoyl cofactor. Knocking out Akr1a1 causes accelerated remodeling of TCA cycle, dysregulated immunoregulatory metabolite level, and altered functional gene expression and cytokine production at later stage of immune response. Thus, the NO*-dependent upregulation of AKR1A1 forms a negative regulatory loop to fine-tune NO*-mediated metabolic and functional remodeling during immune response. Together, this work provided a systems-level map of NO*-dependent regulation, revealed the crosstalk between NO* and immune signaling, and demonstrated mechanisms providing redox adaptation and precise control of NO*s effects.

immunology↗

bioRxiv2020

Redesigning OmpA Loops Using Canonical Outer Membrane Protein Loop Structures

Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test the effect of three different loop types on outer membrane protein A (OmpA) folding. We designed twelve 5-residue loops and experimentally tested the effect of replacing the long loops of outer membrane protein OmpA with the designed loops. Our studies succeeded in creating the smallest known outer membrane barrel. We find that significant changes in OmpA loops do not have a strong overall effect on OmpA folding. However, when decomposing folding into a fast rate and a slow rate we find that changes in loops strongly affect the slow rate of OmpA folding. Extracellular loop types with higher levels of hydrogen bonds had more instances of increasing the slow folding rate and extracellular loop types with low levels of hydrogen bonds had more instances of decreasing the slow folding rate. Having the slow rate affected by loop composition is consistent with the slow rate being associated with the insertion step of outer membrane protein folding.

biophysics↗