Plasma protein signatures of adult asthma.

BACKGROUND: Adult asthma is complex and incompletely understood. Plasma proteomics is an evolving technique that can both generate biomarkers and provide insights into disease mechanisms. We aimed to identify plasma proteomic signatures of adult asthma. METHODS: Protein abundance in plasma was measured in individuals from the Agricultural Lung Health Study (ALHS) (761 asthma, 1095 non-case) and the Atherosclerosis Risk in Communities study (470 asthma, 10,669 non-case) using the SOMAScan 5K array. Associations with asthma were estimated using covariate adjusted logistic regression and meta-analyzed using inverse-variance weighting. Additionally, in ALHS, we examined phenotypes based on both asthma and seroatopy (asthma with atopy (n = 207), asthma without atopy (n = 554), atopy without asthma (n = 147), compared to neither (n = 948)). RESULTS: Meta-analysis of 4860 proteins identified 115 significantly (FDR<0.05) associated with asthma. Multiple signaling pathways related to airway inflammation and pulmonary injury were enriched (FDR<0.05) among these proteins. A proteomic score generated using machine learning provided predictive value for asthma (AUC = 0.77, 95% CI = 0.75-0.79 in training set; AUC = 0.72, 95% CI = 0.69-0.75 in validation set). Twenty proteins are targeted by approved or investigational drugs for asthma or other conditions, suggesting potential drug repurposing. The combined asthma-atopy phenotype showed significant associations with 20 proteins, including five not identified in the overall asthma analysis. CONCLUSION: This first large-scale proteomics study identified over 100 plasma proteins associated with current asthma in adults. In addition to validating previous associations, we identified many novel proteins that could inform development of diagnostic biomarkers and therapeutic targets in asthma management.

Pericentrin deficiency in smooth muscle cells augments atherosclerosis through HSF1-driven cholesterol biosynthesis and PERK activation.

Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) is caused by biallelic loss-of-function variants in pericentrin (PCNT), and premature coronary artery disease (CAD) is a complication of the syndrome. Histopathology of coronary arteries from patients with MOPDII who died of CAD in their 20s showed extensive atherosclerosis. Hyperlipidemic mice with smooth muscle cell-specific (SMC-specific) Pcnt deficiency (PcntSMC-/-) exhibited significantly greater atherosclerotic plaque burden compared with similarly treated littermate controls despite similar serum lipid levels. Loss of PCNT in SMCs induced activation of heat shock factor 1 (HSF1) and consequently upregulated the expression and activity of HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis. The increased cholesterol biosynthesis in PcntSMC-/- SMCs augmented PERK signaling and phenotypic modulation compared with control SMCs. Treatment with the HMGCR inhibitor, pravastatin, blocked the augmented SMC modulation and reduced plaque burden in hyperlipidemic PcntSMC-/- mice to that of control mice. These data support the notion that Pcnt deficiency activates cellular stress to increase SMC modulation and plaque burden, and targeting this pathway with statins in patients with MOPDII has the potential to reduce CAD in these individuals. The molecular mechanism uncovered further emphasizes SMC cytosolic stress and HSF1 activation as a pathway driving atherosclerotic plaque formation independently of cholesterol levels.

Identification of quantitative trait loci for drought tolerance in Bukoba/Kijivu Andean mapping population of common bean.

KEY MESSAGE: Quantitative Trait Loci "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07 and Pv10 of common bean. Drought is a major production constraint of common bean (Phaseolus vulgaris L.) worldwide. The objective of this study was to identify the Quantitative Trait Loci (QTL) for drought tolerance in an Andean population of Recombinant Inbred Lines (RILs). A total of 155 F5:7 RILs derived from a cross between Kijivu (drought tolerant) and Bukoba (drought susceptible) were evaluated for drought tolerance in field and pot experiments. Four field experiments were conducted at three locations in Zambia in 2020 and 2021. All field trials were conducted in the dry season under irrigation. The 155 RILs were genotyped with 11,292 SNPs, and composite interval mapping was conducted to identify QTL for drought tolerance. Seed yield for Kijivu under drought stress was consistently higher than for Bukoba across all four field trials. A total of 60 QTL were identified for morphological, agronomic, and physiological traits under drought stress and non-stress conditions. However, the majority of these QTL were specific to drought stress. QTL "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07, and Pv10. Extensive co-localizations for agronomic and morpho-physiological traits under drought stress were observed at the three drought-tolerance QTL hotspots. Additionally, these three QTL hotspots overlapped with previously identified QTL for drought tolerance, while several others identified QTL are novel. The three identified QTL hotspots could be used in future marker-assisted selection for drought tolerance in common bean.

Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells.

AIMS: The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis. METHODS AND RESULTS: Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice. CONCLUSION: These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.

Smooth Muscle-Alpha Actin R149C Pathogenic Variant Downregulates Integrin Recruitment at Cell-Matrix Adhesions and Decreases Cellular Contractility.

Thoracic aortic aneurysm is found in patients with ACTA2 pathogenic variants. ACTA2 missense variants are associated with impaired aortic smooth muscle cell (SMC) contraction. This study tested the hypothesis that the Acta2R149C/+ variant alters actin isoform expression and decreases integrin recruitment, thus, reducing aortic contractility. Stress relaxation measurements in thoracic aortic rings showed two functional regimes with a reduction of stress relaxation in the aorta from Acta2R149C/+ mice at low tension, but not at high tension values. Contractile responses to phenylephrine and potassium chloride were 50% lower in Acta2R149C/+ mice than in wild-type (WT) mice. Additionally, SMC were immunofluorescently labeled for specific proteins and imaged by confocal or total internal reflection fluorescence microscopy. The quantification of protein fluorescence of Acta2R149C/+ SMC showed a downregulation in smooth muscle α-actin (SMα-actin) and a compensatory upregulation of smooth muscle γ-actin (SMγ-actin) compared to WT cells. These results suggest that downregulation of SMα-actin leads to reduced SMC contractility, while upregulation of SMγ-actin may lead to increased SMC stiffness. Decreased α5ß1 and α2ß1 integrin recruitment at cell-matrix adhesions further reduce the ability of mutant cells to participate in cell-matrix crosstalk. Collectively, the results suggest that mutant Acta2R149C/+ aortic SMC have reduced contractility and interaction with the matrix, which are potential long-term contributing factors to thoracic aortic aneurysms.

Preventing Cholesterol-Induced Perk (Protein Kinase RNA-Like Endoplasmic Reticulum Kinase) Signaling in Smooth Muscle Cells Blocks Atherosclerotic Plaque Formation.

BACKGROUND: Vascular smooth muscle cells (SMCs) undergo complex phenotypic modulation with atherosclerotic plaque formation in hyperlipidemic mice, which is characterized by de-differentiation and heterogeneous increases in the expression of macrophage, fibroblast, osteogenic, and stem cell markers. An increase of cellular cholesterol in SMCs triggers similar phenotypic changes in vitro with exposure to free cholesterol due to cholesterol entering the endoplasmic reticulum, triggering endoplasmic reticulum stress and activating Perk (protein kinase RNA-like endoplasmic reticulum kinase) signaling. METHODS: We generated an SMC-specific Perk knockout mouse model, induced hyperlipidemia in the mice by AAV-PCSK9DY injection, and subjected them to a high-fat diet. We then assessed atherosclerotic plaque formation and performed single-cell transcriptomic studies using aortic tissue from these mice. RESULTS: SMC-specific deletion of Perk reduces atherosclerotic plaque formation in male hyperlipidemic mice by 80%. Single-cell transcriptomic data identify 2 clusters of modulated SMCs in hyperlipidemic mice, one of which is absent when Perk is deleted in SMCs. The 2 modulated SMC clusters have significant overlap of transcriptional changes, but the Perk-dependent cluster uniquely shows a global decrease in the number of transcripts. SMC-specific Perk deletion also prevents migration of both contractile and modulated SMCs from the medial layer of the aorta. CONCLUSIONS: Our results indicate that hypercholesterolemia drives both Perk-dependent and Perk-independent SMC modulation and that deficiency of Perk significantly blocks atherosclerotic plaque formation.

Light potentials of photosynthetic energy storage in the field: what limits the ability to use or dissipate rapidly increased light energy?

The responses of plant photosynthesis to rapid fluctuations in environmental conditions are critical for efficient conversion of light energy. These responses are not well-seen laboratory conditions and are difficult to probe in field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint (Mentha sp.), we show that 'light potentials' for linear electron flow and non-photochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient photosynthetically active radiation (PAR) or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly induced NPQ to photosynthetic control of electron flow at the cytochrome b6f complex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, probably inducing photodamage, revealing a potential target for improving the efficiency and robustness of photosynthesis. We discuss the implications of the approach for open science efforts to understand and improve crop productivity.

Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex.

Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.

Cholesterol-Induced Phenotypic Modulation of Smooth Muscle Cells to Macrophage/Fibroblast-like Cells Is Driven by an Unfolded Protein Response.

OBJECTIVE: Vascular smooth muscle cells (SMCs) dedifferentiate and initiate expression of macrophage markers with cholesterol exposure. This phenotypic switching is dependent on the transcription factor Klf4 (Krüppel-like factor 4). We investigated the molecular pathway by which cholesterol induces SMC phenotypic switching. Approach and Results: With exposure to free cholesterol, SMCs decrease expression of contractile markers, activate Klf4, and upregulate a subset of macrophage and fibroblast markers characteristic of modulated SMCs that appear with atherosclerotic plaque formation. These phenotypic changes are associated with activation of all 3 pathways of the endoplasmic reticulum unfolded protein response (UPR), Perk (protein kinase RNA-like endoplasmic reticulum kinase), Ire (inositol-requiring enzyme) 1α, and Atf (activating transcription factor) 6. Blocking the movement of cholesterol from the plasma membrane to the endoplasmic reticulum prevents free cholesterol-induced UPR, Klf4 activation, and upregulation of the majority of macrophage and fibroblast markers. Cholesterol-induced phenotypic switching is also prevented by global UPR inhibition or specific inhibition of Perk signaling. Exposure to chemical UPR inducers, tunicamycin and thapsigargin, is sufficient to induce these same phenotypic transitions. Finally, analysis of published single-cell RNA sequencing data during atherosclerotic plaque formation in hyperlipidemic mice provides preliminary in vivo evidence of a role of UPR activation in modulated SMCs. CONCLUSIONS: Our data demonstrate that UPR is necessary and sufficient to drive phenotypic switching of SMCs to cells that resemble modulated SMCs found in atherosclerotic plaques. Preventing a UPR in hyperlipidemic mice diminishes atherosclerotic burden, and our data suggest that preventing SMC transition to dedifferentiated cells expressing macrophage and fibroblast markers contributes to this decreased plaque burden.

Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death.

The endoplasmic reticulum, the cytoplasmic organelle that matures a massive amount of nascent secretory polypeptides, is particularly sensitive to stress. Endoplasmic reticulum stress causes unfolded proteins to populate the organelle, eliciting the unfolded protein response. During the unfolded protein response, GRP78-an endoplasmic reticulum master stress regulator-detaches from three endoplasmic reticulum stress sensors (IRE1α, PERK, and ATF6) and allows them to activate the apoptotic signaling pathway. Fortilin, a pro-survival molecule, is known to inhibit apoptosis by binding and inhibiting p53, but its role in endoplasmic reticulum stress-induced apoptosis remains unknown. Here, we report that fortilin directly interacts with the cytoplasmic domain of IRE1α, inhibits both kinase and endoribonuclease (RNase) activities of the stress sensor, and protects cells against apoptotic cell death at both cellular and whole animal levels. Our data support a role of fortilin in the unfolded protein response and its potential participation in human diseases caused by unfolded protein response.IRE1α is an ER stress sensor, whose activity induces apoptosis. Here, the authors report that fortilin, a pro-survival factor, with yet unknown roles in ER stress, interacts with active IRE1α, inhibits both its kinase end RNase activities, and protects cells from apoptosis both in vitro and in vivo.

Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egr-1 Zinc-Finger Protein with DNA.

The transcription factor Egr-1 specifically binds as a monomer to its 9 bp target DNA sequence, GCGTGGGCG, via three zinc fingers and plays important roles in the brain and cardiovascular systems. Using fluorescence-based competitive binding assays, we systematically analyzed the impacts of all possible single-nucleotide substitutions in the target DNA sequence and determined the change in binding free energy for each. Then, we measured the changes in binding free energy for sequences with multiple substitutions and compared them with the sum of the changes in binding free energy for each constituent single substitution. For the DNA variants with two or three nucleotide substitutions in the target sequence, we found excellent agreement between the measured and predicted changes in binding free energy. Interestingly, however, we found that this thermodynamic additivity broke down with a larger number of substitutions. For DNA sequences with four or more substitutions, the measured changes in binding free energy were significantly larger than predicted. On the basis of these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of such sequences that might functionally sequester Egr-1.

NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups.

NMR scalar couplings across hydrogen bonds represent direct evidence for the partial covalent nature of hydrogen bonds and provide structural and dynamic information on hydrogen bonding. In this article, we report heteronuclear 15N-31P and 1H-31P scalar couplings across the intermolecular hydrogen bonds between protein histidine (His) imidazole and DNA phosphate groups. These hydrogen-bond scalar couplings were observed for the Egr-1 zinc-finger-DNA complex. Although His side-chain NH protons are typically undetectable in heteronuclear 1H-15N correlation spectra due to rapid hydrogen exchange, this complex exhibited two His side-chain NH signals around 1H 14.3 ppm and 15N 178 ppm at 35 °C. Through various heteronuclear multidimensional NMR experiments, these signals were assigned to two zinc-coordinating His side chains in contact with DNA phosphate groups. The data show that the Nδ1 atoms of these His side chains are protonated and exhibit the 1H-15N cross-peaks. Using heteronuclear 1H, 15N, and 31P NMR experiments, we observed the hydrogen-bond scalar couplings between the His 15Nδ1/1Hδ1 and DNA phosphate 31P nuclei. These results demonstrate the direct involvement of the zinc-coordinating His side chains in the recognition of DNA by the Cys2His2-class zinc fingers in solution.

Regulation of transcription factors via natural decoys in genomic DNA.

Eukaryotic genomic DNA contains numerous high-affinity sites for transcription factors. Only a small fraction of these sites directly regulates target genes. Other high-affinity sites can serve as naturally present decoys that sequester transcription factors. Such natural decoys in genomic DNA may provide novel regulatory mechanisms for transcription factors.

Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling.

The residence times of molecular complexes in solution are important for understanding biomolecular functions and drug actions. We show that NMR data of intermolecular hydrogen-bond scalar couplings can yield information on the residence times of molecular complexes in solution. The molecular exchange of binding partners via the breakage and reformation of a complex causes self-decoupling of intermolecular hydrogen-bond scalar couplings, and this self-decoupling effect depends on the residence time of the complex. For protein-DNA complexes, we investigated the salt concentration dependence of intermolecular hydrogen-bond scalar couplings between the protein side-chain (15)N and DNA phosphate (31)P nuclei, from which the residence times were analyzed. The results were consistent with those obtained by (15)Nz-exchange spectroscopy. This self-decoupling-based kinetic analysis is unique in that it does not require any different signatures for the states involved in the exchange, whereas such conditions are crucial for kinetic analyses by typical NMR and other methods.

Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice.

Fortilin, a pro-survival molecule, inhibits p53-induced apoptosis by binding to the sequence-specific DNA-binding domain of the tumor suppressor protein and preventing it from transcriptionally activating Bax. Intriguingly, fortilin protects cells against ROS-induced cell death, independent of p53. The signaling pathway through which fortilin protects cells against ROS-induced cell death, however, is unknown. Here we report that fortilin physically interacts with the antioxidant enzyme peroxiredoxin-1 (PRX1), protects it from proteasome-mediated degradation, and keeps it enzymatically active by blocking its deactivating phosphorylation by Mst1, a serine/threonine kinase. At the whole animal level, the liver-specific overexpression of fortilin reduced PRX1 phosphorylation in the liver, enhanced PRX1 activity, and protected the transgenic animals against alcohol-induced, ROS-mediated, liver damage. These data suggest the presence of a novel oxidative-stress-handling pathway where the anti-p53 molecule fortilin augments the peroxidase PRX1 by protecting it against degradation and inactivation of the enzyme. Fortilin-PRX1 interaction in the liver could be clinically exploited further to prevent acute alcohol-induced liver damage in humans.