Genetic Attenuation of Paraoxonase 1 Activity Induces Proatherogenic Changes in Plasma Proteomes of Mice and Humans

High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, possesses anti-inflammatory, antioxidative, and antithrombotic activities. Paraoxonase 1 (PON1), carried on HDL in the blood, can contribute to these antiatherogenic activities. The PON1-Q192R polymorphism involves a change from glutamine (Q variant) to arginine (R variant) at position 192 of the PON1 protein and affects its enzymatic activity. The molecular basis of PON1 association with cardiovascular and neurological diseases is not fully understood. To get insight into the function of PON1 in human disease, we examined how genetic attenuation of PON1 levels/activity affect plasma proteomes of mice and humans. Healthy participants (48.9 years old, 50% women) were randomly recruited from the Poznań population. Four-month-old Pon1−/− (n = 17) and Pon1+/+ (n = 8) mice (50% female) were used in these experiments. Plasma proteomes were analyzed using label-free mass spectrometry. Bioinformatics analysis was carried out using the Ingenuity Pathway Analysis (IPA) resources. PON1-Q192R polymorphism and Pon1−/− genotype induced similar changes in plasma proteomes of humans and mice, respectively. The top molecular network, identified by IPA, affected by these changes involved proteins participating in lipoprotein metabolism. Other PON1 genotype-dependent proteomic changes affect different biological networks in humans and mice: “cardiovascular, neurological disease, organismal injury/abnormalities” in PON1-192QQ humans and “humoral immune response, inflammatory response, protein synthesis” and “cell-to-cell signaling/interaction, hematological system development/function, immune cell trafficking” in Pon1−/− mice. Our findings suggest that PON1 interacts with molecular pathways involved in lipoprotein metabolism, acute/inflammatory response, and complement/blood coagulation that are essential for blood homeostasis. Modulation of those interactions by the PON1 genotype can account for its association with cardiovascular and neurological diseases.


Introduction
Inflammation promotes several key events during the development of atherosclerosis. High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, has also been reported to possess anti-inflammatory, antioxidative, and antithrombotic activities important for its atheroprotective function [1]. A calcium-dependent hydrolytic enzyme, paraoxonase 1 (PON1),

Data Analysis
Datasets were imported into MaxQuant 1.5.3.30 version. Proteins were identified using UniProt human/mouse database with a precision tolerance 10 ppm for peptide masses and 0.08 Da for fragment ion masses as previously described [25]. The MaxQuant data were filtered for reverse identifications (false positives), contaminants, and proteins "only identified by site". Perseus software (version 1.4.1.3, MPIB, Martinsried, Germany) was used for label-free quantification (LFQ) of peptide intensities. The mean LFQ intensities +/standard deviation were calculated for each identified differentially expressed protein. The fold change values (FC) for the PON1 genotype-affected proteins were calculated from the mean LFQ intensities for each genotype.

Statistics
Proteins were considered to be PON1 genotype-dependent if at least two peptides with >99% confidence were identified and the FC between PON1 genotype groups was statistically significant (p < 0.05). Numeric data were log-transformed and filtered. For multiple comparisons, one-way analysis of variance (ANOVA) with a Bonferroni correction for multiple testing was carried out. PON1 genotype-affected proteins were normalized using Z-score algorithm. T-test was used for Antioxidants 2020, 9, 1198 4 of 17 comparisons between two groups with p values < 0.05 considered significant. Multivariate analyses were carried out by untargeted principal component analysis (PCA) [25].

Bioinformatics Analysis
The datasets containing differentially expressed proteins were uploaded into the IPA Knowledge database. Biological pathways and networks involving PON1 genotype-affected proteins were identified using the Ingenuity Pathway Analysis resources (IPA, Ingenuity Systems, Mountain View, CA, USA) as previously described [25].

Results
The effects of the PON1 genotype on PON1 activity and protein levels in humans and mice are shown in Table 1. As expected, deletion of the Pon1 gene had a severe effect on Pon1 protein and activity levels in mice, with essentially complete absence of both Pon1 protein and activity [6,13,22]. In contrast, the PON1-Q192R polymorphism on PON1 function in humans was relatively mild with about 40% reduction in PON1 protein and fivefold reduction in PON1 activity. In each group of human participants and mice, label-free nanoLC-MS/MS mass spectrometry identified 196-198 plasma proteins with a minimum of two peptides and 1% false discovery rate (FDR). Proteome Discoverer (PD) analysis showed >90% overlap at the protein level between duplicate runs.
The variation between samples in terms of global proteomic profiles was assessed using the principal component analysis (PCA). There was a clear difference in PCA profiles between Pon1 −/− mice and their Pon1 +/+ siblings ( Figure 1A). However, there was an overlap in PCA profiles between humans with PON1-192QQ, PON1-192QR, and PON1-192RR genotypes ( Figure 1B). Antioxidants 2020, 9, x FOR PEER REVIEW 4 of 17 comparisons between two groups with p values < 0.05 considered significant. Multivariate analyses were carried out by untargeted principal component analysis (PCA) [25].

Bioinformatics Analysis
The datasets containing differentially expressed proteins were uploaded into the IPA Knowledge database. Biological pathways and networks involving PON1 genotype-affected proteins were identified using the Ingenuity Pathway Analysis resources (IPA, Ingenuity Systems, Mountain View, CA, USA) as previously described [25].

Results
The effects of the PON1 genotype on PON1 activity and protein levels in humans and mice are shown in Table 1. As expected, deletion of the Pon1 gene had a severe effect on Pon1 protein and activity levels in mice, with essentially complete absence of both Pon1 protein and activity [6,13,22]. In contrast, the PON1-Q192R polymorphism on PON1 function in humans was relatively mild with about 40% reduction in PON1 protein and fivefold reduction in PON1 activity. In each group of human participants and mice, label-free nanoLC-MS/MS mass spectrometry identified 196-198 plasma proteins with a minimum of two peptides and 1% false discovery rate (FDR). Proteome Discoverer (PD) analysis showed >90% overlap at the protein level between duplicate runs.
The variation between samples in terms of global proteomic profiles was assessed using the principal component analysis (PCA). There was a clear difference in PCA profiles between Pon1 −/− mice and their Pon1 +/+ siblings ( Figure 1A). However, there was an overlap in PCA profiles between humans with PON1-192QQ, PON1-192QR, and PON1-192RR genotypes ( Figure 1B). We identified 50 differentiating proteins affected by the Pon1 −/− genotype in mice and 21 differentiating proteins affected by the PON1-Q192R polymorphism in humans ( Figure 2). Of these, 41 proteins (84%) were affected only in mice, while 12 proteins (57%) were affected only in humans. There were nine proteins, accounting for 43% and 22% of the differentiating proteins in humans and mice, respectively, that were affected by the PON1 genotype in both species (Figure 2). The differentiating proteins and their functions are listed in Supplementary Table S2.

Overlap between Proteins Affected by PON1 Genotype in Humans and Mice
We identified nine proteins whose levels were affected both by the PON1-Q192R polymorphism in humans and the Pon1 −/− genotype in mice ( Figure 2, Table 2). The nine shared proteins represented 43% and 18% of the total number of proteins affected by PON1 genotype in humans and mice, respectively. Of those common proteins, five were affected in the same direction in humans and mice (i.e., were either upregulated (APOD, APOM) or downregulated (APOA1, F13B, PON1) in both species). Four other proteins were affected in a different direction in humans and mice: FETUB, HP, and TTR were downregulated in humans and upregulated in mice, while IGHG3 was upregulated in humans and downregulated in mice.

Bioinformatics Analysis
To identify biological pathways linked to proteins affected by the PON1 genotype in humans and mice, bioinformatics analysis with IPA resources was carried out. We found that proteins affected by the human PON1-Q192R polymorphism were significantly enriched in 11 canonical pathways, which are linked to atherosclerosis, thrombosis, and Alzheimer's disease ( Figure 4). Proteins in 10 of those pathways were also significantly enriched in Pon1 −/− mice.
Fifteen other pathways contained proteins significantly enriched only in Pon1 −/− mice and affected glucose/energy metabolism, iron homeostasis, and immune system.

Human PON1-Q192R Polymorphism
IPA identified two top-scoring biological networks associated with human PON1-Q192R polymorphism: "Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry" and "Cardiovascular Disease, Neurological Disease, Organismal Injury and Abnormalities" (Table 3). Table 3. Top molecular networks associated with human PON1-Q192R polymorphism. Upregulated (↑) and downregulated (↓) proteins are highlighted in bold red and green, respectively. Graphical illustrations of interactions between proteins in these networks are shown in Figure 5. The "lipid metabolism, molecular transport, small molecule biochemistry" network, identified from analyses PON1-192QQ vs. QR+RR, had a score of 27 and contained 35 molecules, including 27 proteins. Nine proteins from this network were quantified by label-free mass spectrometry, while 17 proteins and 9 other molecules were identified by IPA to interact with the quantified proteins (Table 3). Graphical illustration of this network is shown in Figure 5A. Proteins in this network participate in lipid metabolism and acute phase/immune response and show strong interactions centering on the lipoproteins LDL and PON1/HDL, the cytokine IL6, and TGFB1. Similar lipid metabolism networks were identified from analyses PON1-PON1-192QQ vs. QR and PON1-192QQ vs. RR ( Table 3), suggesting that this network was associated with PON1-192QQ polymorphism.

Analysis
The "cardiovascular disease, neurological disease, organismal injury and abnormalities" network, identified from analysis PON1-192QQ vs. RR, was associated with PON1-192RR polymorphism. The cardiovascular/neurological disease network has a score of 14 and contains 35 molecules, including 31 proteins. Five proteins from this network were quantified by label-free mass spectrometry while 26 proteins and 4 other molecules were identified by IPA to interact with the quantified proteins ( Table 3). Proteins of this network participate in oxidative stress response, lipoprotein metabolism, and NF-κB signaling and show strong interactions centering on the NF-κB inhibitor alpha NFKBIA and HDL ( Figure 5B). lipoprotein metabolism, and NF-κB signaling and show strong interactions centering on the NF-κB inhibitor alpha NFKBIA and HDL ( Figure 5B). A. B.

Pon1 −/− Mouse Genotype
IPA identified three top-scoring biological networks associated with mouse Pon1 −/− genotype ( Table 4). The "Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry" network, with a score of 41, contains 32 proteins. Eighteen of those proteins were quantified by label-free mass spectrometry while 14 were identified by IPA to interact with the quantified proteins. This network involves proteins participating in lipid metabolism, iron metabolism, acute phase response, and complement/coagulation cascades that show strong interactions centering on lipoproteins PON1/APOA1/HDL and APOB/LDL, and ERK1/2 ( Figure 6A). Table 4. Top molecular networks associated with Pon1 −/− genotype in mice. Upregulated (↑) and downregulated (↓) proteins are highlighted in bold red and green, respectively. Graphical illustrations of interactions between proteins in these networks are shown in Figure 6A-C.

Analysis
Molecules in Network Score Focus Molecules

Top Diseases and Functions
Pon1 −/− vs. Pon1 +/+ Figure 6A    Cell-to-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking The "Humoral Immune Response, Inflammatory Response, Protein Synthesis" network with a score of 26 contains 36 proteins. Twelve of those proteins were quantified by label-free mass spectrometry while 25 were identified by IPA to interact with the quantified proteins. This network contains immunoglobulins and other proteins participating in the acute phase/immune response, which show strong interactions centering on ERK, P38/MAPK, Akt, and NF-κB ( Figure 6B).
The "Cell-to-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking" network with a score of 26 contains 33 proteins. Twelve of those proteins were quantified by label-free mass spectrometry while 21 were identified by IPA to interact with the quantified proteins. This network involves proteins participating in cell signaling and acute phase/immune response, which show strong interactions centering on Tgfb1, Vegf, Jnk ( Figure 6C).   The "Humoral Immune Response, Inflammatory Response, Protein Synthesis" network with a score of 26 contains 36 proteins. Twelve of those proteins were quantified by label-free mass spectrometry while 25 were identified by IPA to interact with the quantified proteins. This network contains immunoglobulins and other proteins participating in the acute phase/immune response, which show strong interactions centering on ERK, P38/MAPK, Akt, and NF-κB ( Figure 6B).
The "Cell-to-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking" network with a score of 26 contains 33 proteins. Twelve of those proteins were quantified by label-free mass spectrometry while 21 were identified by IPA to interact with the quantified proteins. This network involves proteins participating in cell signaling and acute phase/immune response, which show strong interactions centering on Tgfb1, Vegf, Jnk ( Figure 6C).

Discussion
The present study provides evidence that genetic reduction of PON1 levels induces proatherogenic changes in plasma proteomes of humans and mice. Specifically, we show for the first time that (i) PON1-Q192R polymorphism in humans and Pon1 −/− genotype in mice induce similar changes in the plasma proteome, which affect a major biological network involving proteins participating in lipoprotein metabolism, the "Lpid Metabolism, Molecular Transport, Small Molecule Biochemistry" network; (ii) these genetic variants also induce other changes in plasma proteomes, which are species-specific and affect different biological networks in humans and mice: "Cardiovascular Disease, Neurological Disease, Organismal Injury and Abnormalities" in PON1-192QQ vs. RR humans and "Humoral Immune Response, Inflammatory Response, Protein Synthesis" and "Cell-to-Cell Signaling and Interaction, Hematological System Development and

Discussion
The present study provides evidence that genetic reduction of PON1 levels induces proatherogenic changes in plasma proteomes of humans and mice. Specifically, we show for the first time that (i) PON1-Q192R polymorphism in humans and Pon1 −/− genotype in mice induce similar changes in the plasma proteome, which affect a major biological network involving proteins participating in lipoprotein metabolism, the "Lpid Metabolism, Molecular Transport, Small Molecule Biochemistry" network; (ii) these genetic variants also induce other changes in plasma proteomes, which are species-specific and affect different biological networks in humans and mice: "Cardiovascular Disease, Neurological Disease, Organismal Injury and Abnormalities" in PON1-192QQ vs. RR humans and "Humoral Immune Response, Inflammatory Response, Protein Synthesis" and "Cell-to-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking" in Pon1 −/− vs. Pon1 +/+ mice. Comparative proteomics of the human PON1-Q192R polymorphism and the mouse Pon1 −/− genotype have not been examined before, and, to our best knowledge, this is the first study of plasma proteomes in PON1-Q192R humans and Pon1 −/− mice. Overall, these changes in plasma proteomes are proatherothrombotic and are known to be associated with human cardiovascular and neurological diseases.
Some of the plasma proteins affected by the PON1 genotype are shared between humans and mice while changes in other proteins are species-specific and limited to humans or mice. The shared proteins include lipoproteins APOD and APOM (negative regulators of lipoprotein oxidation), the carrier of hydrophobic molecules AFM (involved in transport of fatty acids, or vitamin E when the lipoprotein system is insufficient), the negative regulator of endopeptidase activity FETUB, the antioxidant HP (involved in acute inflammatory response), the thyroid hormone-binding protein TTR (involved in thyroxine transport from the blood to the brain), the fibrin-stabilizing factor F13B (associated with a risk of stroke), and the immunoglobulin IGHG3 (involves B-cell signaling, complement activation, and humoral immunity). The species-specific proteins affected by the PON1 genotype are more numerous in mice (n = 41) than in humans (n = 12) and most likely reflect a much more severe effect of the Pon1 gene deletion on Pon1 function (essentially complete absence of both Pon1 protein and activity) in mice [6,13,22] than the effect of PON1-Q192R polymorphism on PON1 function in humans (about 40% reduction in PON1 protein and 10-fold in PON1 activity).
We found that the lipoproteins APOD and APOM were upregulated by PON1-192QR vs. PON1-192RR genotype in humans and Pon1 −/− genotype in mice. Elevation in APOD expression has been associated with a number of pathological conditions including neurodegenerative disease [26]. Both APOD and APOM are negative regulators of lipoprotein oxidation and have antiatherogenic properties. ApoD is an acute response protein with a protective function mediated by the control of peroxidized lipids. In mice, deletion of the ApoD gene increases the sensitivity to oxidative stress, levels of lipid peroxides in the brain, and impairs learning and locomotor abilities [26]. APOM binds oxidized phospholipids in plasma and increases the antioxidant effect of HDL [27]. Thus, changes in APOD and APOM levels could contribute to the association of the PON1 genotype with cardiovascular and neurological diseases.
In the present study we found that ApoA1 and ApoC1, known to be negative regulators of lipid/cholesterol catabolism/transport cholesterol, were downregulated, while ApoB, a positive regulator of lipid/cholesterol storage, was upregulated in Pon1 −/− mice. These findings suggest that the Pon1 −/− genotype exerts a proatherogenic effect on the lipoprotein homeostasis.
We identified several proteins of the coagulation system that were affected by the PON1 genotype. For example, PLG, SERPINA10, and F13B were downregulated by the PON1-192QQ genotype in humans. PLG, bound to fibrin clots, is converted to plasmin, which plays a crucial role in dissolving blood clots. SERPINA1, a negative regulator of blood coagulation, inhibits the activated coagulation factors X and XI, thus preventing fibrin clot formation [28]. F13B is a subunit of prototransglutaminase, which is activated at the final stage of coagulation and which stabilizes the clot by forming an amide bond between Glu and Lys residues of fibrin [29]. A genetic variant of F13B is associated with the risk of stroke [30]. Thus, reduced levels of PLG, SERPINA10, and F13B in carriers of PON1-192QQ genotype suggest that the 192QQ variant could increase the propensity to atherothrombosis, which might account for an association of this variant with vascular disease [16,31].
The antithrombin Serpinc1, a component of the complement/coagulation cascades, was downregulated in Pon1 −/− mice. As genetic variants of SERPINC1 are known to be associated with venous thrombosis in humans [32], the downregulation of the antithrombin Serpinc1 in Pon1 −/− mice could increase thrombin activity, thereby increasing blood clotting, which might account for increased atherosclerotic lesions observed in these animals when fed with a high-fat diet [13].

Conclusions
Our findings identify a proatherogenic phenotype in the plasma proteome associated with the PON1 genotype in humans and mice. This phenotype includes mild dysregulation of lipoproteins but is silent, even in Pon1 −/− mice. However, it can be exacerbated by a stress such as severe dyslipidemia induced by a proatherogenic high-fat diet. Indeed, Pon1 −/− mice became susceptible to aortic lesions only when fed with a high-fat diet but not a standard chow diet [13]. Other metabolic stressors, such as elevated Hcy, have been found to modulate expression of brain proteins involved in oxidative stress and neurodegeneration in Pon1 −/− mice [34].