Decoding the Molecular Universe -- Workshop Report

On August 9-10, 2023, a workshop was convened at the Pacific Northwest National Laboratory (PNNL) in Richland, WA that brought together a group of internationally recognized experts in metabolomics, natural products discovery, chemical ecology, chemical and biological threat assessment, cheminformatics, computational chemistry, cloud computing, artificial intelligence, and novel technology development. These experts were invited to assess the value and feasibility of a grand-scale project to create new technologies that would allow the identification and quantification of all small molecules, or to decode the molecular universe. The Decoding the Molecular Universe project would extend and complement the success of the Human Genome Project by developing new capabilities and technologies to measure small molecules (defined as non-protein, non-polymer molecules less than 1500 Daltons) of any origin and generated in biological systems or produced abiotically. Workshop attendees 1) explored what new understanding of biological and environmental systems could be revealed through the lens of small molecules; 2) characterized the similarities in current needs and technical challenges between each science or mission area for unambiguous and comprehensive determination of the composition and quantities of small molecules of any sample; 3) determined the extent to which technologies or methods currently exist for unambiguously and comprehensively determining the small molecule composition of any sample and in a reasonable time; and 4) identified the attributes of the ideal technology or approach for universal small molecule measurement and identification. The workshop concluded with a discussion of how a project of this scale could be undertaken, possible thrusts for the project, early proof-of-principle applications, and similar efforts upon which the project could be modeled

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H+ Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events

Increasing frequency and duration of Arctic winter warming events

Near-surface air temperatures close to 0°C were observed in situ over sea ice in the central Arctic during the last three winter seasons. Here we use in situ winter (December–March) temperature observations, such as those from Soviet North Pole drifting stations and ocean buoys, to determine how common Arctic winter warming events are. Observations of winter warming events exist over most of the Arctic Basin. Temperatures exceeding -5°C were observed during >30% of winters from 1954 to 2010 by North Pole drifting stations or ocean buoys. Using the ERA-Interim record (1979–2016), we show that the North Pole (NP) region typically experiences 10 warming events (T2m > 10°C) per winter, compared with only five in the Pacific Central Arctic (PCA). There is a positive trend in the overall duration of winter warming events for both the NP region (4.25 days/decade) and PCA (1.16 days/decade), due to an increased number of events of longer duration

Myosin Light Chain Kinase Signaling in Endothelial Barrier Dysfunction

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability

Interleukin-1β-induced barrier dysfunction is signaled through PKC-θ in human brain microvascular endothelium

Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor Gö6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by Gö6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions

Rapid loss of blood–brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay

Blood–brain barrier (BBB) P-glycoprotein activity is rapidly reduced by vascular endothelial growth factor (VEGF) acting via Src and by tumor necrosis factor-α acting via protein kinase C (PKC)β1. To probe underlying mechanism(s), we developed an in vivo, immunoblot-based proteinase K (PK) protection assay to assess the changes in the P-glycoprotein content of the BBB's luminal membrane. Infusion of PK into the brain vasculature selectively cleaved luminal membrane P-glycoprotein, leaving intracellular proteins intact. Intracerebroventricular injection of VEGF partially protected P-glycoprotein from proteolytic cleavage, consistent with transporter internalization. Activation of PKCβ1 did not protect P-glycoprotein. Thus, VEGF and PKCβ1 reduce P-glycoprotein activity by distinct mechanisms

Interleukin-1β-Induced Barrier Dysfunction Is Signaled Through Pkc-θ in Human Brain Microvascular Endothelium

Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor Gö6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by Gö6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions. brain inflammation is a pathological consequence of trauma, stroke, cerebral infection, multiple sclerosis, and other inflammatory conditions (4, 10, 30, 34). A serious consequence of brain inflammation is microvascular leakage and brain edema, leading to brain swelling, neuronal injury, and death. Microvascular leakage occurs due to misregulation of the protective interface between the blood and the brain tissue known as the blood-brain barrier (BBB; for review see Refs. 1, 12, 17, 22). During brain inflammation, microvascular barriers become compromised in response to changes in expression or organization of endothelial tight junction proteins permitting plasma components to leak across the BBB into the brain tissue interstitial space. This BBB hyperpermeability occurs in response to proinflammatory agents that are present in the brain tissue during inflammation. The proinflammatory cytokine interleukin (IL)-1β is produced in the brain during neuroinflammation and contributes to brain microvascular leakage and brain edema (8, 10, 13, 30, 34). The proedematous effects of IL-1β are mediated through the IL-1 receptor (IL1-R1), in that direct inhibition of IL-1β binding to IL1-R1 prevented both brain edema and brain tissue injury in rodent models of experimental cerebral ischemia (38). Brain edema resulting from hypoxic/ischemic injury was also prevented in IL1-R1 knockout mice compared with wild-type mice (27). In addition, vascular effects result from direct exposure of endothelial cells to IL-1β, as demonstrated in studies (14) with cultured human brain microvascular endothelial cells where IL-1β treatment induced hyperpermeability in the absence of all other brain parenchymal cell types. While the effects of IL-1β on brain endothelial hyperpermeability have been clearly demonstrated, most of the intermediate cellular signaling events leading to hyperpermeability are unknown. In many tissue types, microvascular hyperpermeability is signaled through classical (α, βI, βII, and γ), novel (δ, ε, η, θ, and μ), or atypical (ζ and λ/ι) protein kinase C (PKC) isoforms (32, 39–41). For example, microvascular leakage was mediated by PKC-βII (2, 3), -δ (25), and -ζ (6) at the blood-retinal barrier. Likewise, the isoform-selective PKC inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] prevented microvascular leakage during ischemia-reperfusion injury in the rat heart, as well as hyperpermeability in human coronary microvascular endothelium in response to IL-1β exposure (36). In addition, BBB dysfunction was mediated by PKC-α in response to tumor necrosis factor (TNF)-α exposure in mouse brain endothelium (29) and involved activation of PKC-θ and possibly other PKC isoforms during hypoxia in the rat brain (18, 37). Based on these observations, we hypothesized that select PKC isoforms mediate hyperpermeability in human brain microvascular endothelium during IL-1β exposure. In the present study, we examined the role of PKC in mediating barrier dysfunction in human brain microvascular endothelium in response to IL-1β exposure. Our data indicated that novel PKC isoforms (δ, θ, and μ) were activated in response to IL-1β exposure in human brain microvascular endothelial cells (hBMECs). Evidence from PKC isoform-specific inhibitors and gene silencing indicated that PKC-θ was necessary for IL-1β-induced barrier dysfunction [measured as decreased transendothelial electrical resistance (TER)] in hBMEC monolayers. We found that decreased TER was not accompanied by altered expression of junction proteins, increased cell contractility, or apoptotic mechanisms. Furthermore, immunoprecipitation experiments demonstrated that decreased TER in response to IL-1β involved PKC-θ-dependent phosphorylation of zona occludens (ZO)-1. Therefore, selective inhibition of PKC-θ under inflammatory conditions may prevent BBB leakage that is due to posttranslational modification of tight junction proteins and conformational modification of tight junctions in the brain microvascular endothelium