Understanding the Role of Caveolae in Oxygen Buffering: The Effect of Membrane Curvature.

The "oxygen paradox" can be explained as two opposing biological processes with oxygen (O2) as a reactant. On the one hand, oxygen is essential to aerobic metabolism, powering oxidative phosphorylation in mitochondria. On the other hand, an excess supply of oxygen will generate reactive species which are harmful for the cell. In healthy tissues, the first process must be maximized relative to the second one. We have hypothesized that curved and cholesterol-enriched membrane invaginations called caveolae help maintain the proper oxygen level by taking up oxygen and attenuating its release to the mitochondria. The mechanism by which caveolae may help to buffer the oxygen level in cells is still unclear. Here, we aim to assess how structural aspects of caveolae, the curvature of the membrane, influence the local oxygen abundance and the membrane partitioning. We have modelled a flat bilayer and a liposome composed of dipalmitoylphosphatidylcholine (DPPC), using molecular dynamics simulation. Associated changes in the membrane-level oxygen partition coefficient and free energy profiles will be presented.

Do Vascular and Extracellular Measurements Consistently Reflect Intracellular pO2?

Oxygen measurements are routinely made either in the vasculature or in the extracellular fluid surrounding the cells of tissues. Yet, metabolic oxygen availability depends on the pO2 within the cells, as does the enhancing effect of oxygen on radiotherapy outcomes. This article reports quantitative modeling work examining the effect of cellular plasma membrane composition on tissue permeability, as a window into tissue oxygen gradients. Previous application of the model indicates that lipid-mediated diffusion pathways accelerate oxygen transfer from capillaries to intracellular compartments and that the extent of acceleration is modulated by membrane lipid and protein composition. Here, the effects of broken intercellular junctions and increased gap size between cells in the model are addressed. The conclusion is reached that the pO2 gradient will likely be consistent among similar, healthy tissues but may increase with increased interstitial fluid fraction and broken intercellular junctions. Therefore, tissue structural changes in tumors and other diseased or damaged tissues may lead to aberrations in permeability that confound interpretation of extracellular oxygen measurements.

Correction to: Atomistic Simulations Modify Interpretation of Spin-Label Oximetry Data. Part 1: Intensified Water-Lipid Interfacial Resistances.

[This corrects the article PMC10249954.].

Simulation Study of Breast Cancer Lipid Changes Affecting Membrane Oxygen Permeability: Effects of Chain Length and Cholesterol.

Tumor radiotherapy relies on intracellular oxygen (O2) to generate reactive species that trigger cell death, yet hypoxia is common in cancers of the breast. De novo lipid synthesis in tumors supports cell proliferation but also may lead to unusually high levels of the 16:1 palmitoleoyl (Y) phospholipid tail, which is two carbons shorter than the 18:1 oleoyl (O) tail abundant in normal breast tissue. Here, we use atomic resolution molecular dynamics simulations to test two hypotheses: (1) the shorter, 16:1 Y, tail of the de novo lipid biosynthesis product 1-palmitoyl,2-palmitoleoyl-phosphatidylcholine (PYPC) promotes lower membrane permeability relative to the more common lipid 1-palmitoyl,2-oleoylphosphatidylcholine (POPC), by reducing oxygen solubility in the interleaflet region, and (2) cholesterol further lessens the permeability of PYPC by reducing overall O2 solubility and promoting PYPC tail order adjacent to the rigid cholesterol ring system. The simulations conducted here indicate that PYPC has a permeability of 14 ± 1 cm/s at 37 °C, comparable to 15.4 ± 0.4 cm/s for POPC. Inclusion of cholesterol in a 1:1 ratio with phospholipid intensifies the effect of chain length, giving permeabilities of 10.2 ± 0.2 cm/s for PYPC/cholesterol and 11.0 ± 0.6 cm/s for POPC/cholesterol. These findings indicate that PYPC may not substantially influence membrane-level oxygen flux and is unlikely to hinder breast tissue oxygenation.

Updated Evaluation of Cholesterol's Influence on Membrane Oxygen Permeability.

There is a surprising gap in knowledge regarding the mechanism of oxygen (O2) diffusional delivery at the level of tissues and cells. Yet, the effectiveness of tumor radiotherapy, the success of tissue engineering, and healthy metabolism all require ample intracellular oxygen. Tissue-level diffusion takes place in a complex and crowded macromolecular environment. Cholesterol-rich cellular membranes have been thought to reduce oxygen flux. Here, we use atomistic molecular dynamics simulations to update prior estimates of bilayer permeability and related parameters for 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers, using a modified O2 model with improved membrane-water partitioning behavior. This work estimates an oxygen permeability coefficient of 15 ± 1 cm/s for POPC and 11.5 ± 0.4 cm/s for POPC/cholesterol (1:1 molecular ratio) at 37 °C. The permeability of POPC is found to be ~1/3 that of a water layer of similar thickness, and the permeability of POPC/cholesterol is estimated to be 20-30% below that of POPC. Void pathway visualization and free energy data support channeling of oxygen toward the center of cholesterol-incorporating membranes, while partition coefficient data suggest reduced membrane solubility of oxygen due to cholesterol. Further study is needed to understand whether diffusion pathway changes due to cholesterol and other molecular compositional factors influence oxygen availability within tissue.

Discerning Membrane Steady-State Oxygen Flux by Monte Carlo Markov Chain Modeling.

Molecular oxygen (O2) permeability coefficients for lipid bilayers have previously been estimated using both electron paramagnetic resonance (EPR) oximetry and molecular dynamics simulation data. Yet, neither technique captures the fluxes that exist physiologically. Here, the dynamic steady state is modeled using a stochastic approach built on atomic resolution molecular dynamics simulation data. A Monte Carlo Markov chain technique is used to examine membrane-level fluxes of oxygen in lipid-water systems. At steady state, the concentration of oxygen is found to be higher inside the model membranes than in surrounding water, consistent with the known favorable partitioning of O2 toward the lipid phase. Pure phospholipid 1-palmitoyl,2-oleoyl-phosphatidylcholine (POPC) bilayers accrue ~40% more O2 molecules at steady state than POPC/cholesterol bilayers (1:1 molecular ratio) mimicking the red blood cell membrane. Steady-state levels of oxygen were reached inside both bilayer types within the same timeframe, but depletion of oxygen from the bilayer interior occurred 17% faster for POPC than for POPC/cholesterol. Likewise, first-order rate constants estimated for accrual to steady state were the same for POPC and POPC/cholesterol, at 190 µs-1, while first-order rate constants for depletion of the accrued O2 from the bilayers differed, at 95 µs-1 for POPC and 81 µs-1 for POPC/cholesterol (lower by 15%). These results are consistent with prior experiments in red blood cells (RBCs) with varying membrane cholesterol content, in which additional cholesterol slowed oxygen uptake and release. Further work is needed to understand whether differences in RBC membrane cholesterol content would affect the delivery of oxygen to tissues.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

How does oxygen diffuse from capillaries to tissue mitochondria? Barriers and pathways.

Timely delivery of oxygen (O2 ) to tissue mitochondria is so essential that elaborate circulatory systems have evolved to minimize diffusion distances within tissue. Yet, knowledge is surprisingly limited regarding the diffusion pathway between blood capillaries and tissue mitochondria. An established and growing body of work examines the influence cellular and extracellular structures may have on subcellular oxygen availability. This brief review discusses the physiological and pathophysiological significance of oxygen availability, highlights recent computer modelling studies of transport at the cell-membrane level, and considers alternative diffusion pathways within tissue. Experimental and computer modelling studies suggest that oxygen diffusion may be accelerated by cellular lipids, relative to cytosolic and interstitial fluids. Such acceleration, or 'channelling', would occur due to greatly enhanced oxygen solubility in lipids, especially near the midplane of lipid bilayers. Rapid long-range movement would be promoted by anisotropically enhanced lateral diffusion of oxygen along the midplane and by junctions holding lipid structures in close proximity to one another throughout the tissue. Clarifying the biophysical mechanism of oxygen transport within tissue will shed light on limitations and opportunities in tumour radiotherapy and tissue engineering.

Pathways of Oxygen Diffusion in Cells and Tissues : Hydrophobic Channeling via Networked Lipids.

Oxygen delivery to tissue mitochondria relies on simple diffusion in the target cells and tissues. As such, intracellular availability of O2 in tissue depends on its solubility and diffusivity in complex and heterogeneous macromolecular environments. The path of oxygen diffusion is key to its rate of transfer, especially where pathways of differing favorability are present. Most commonly, aqueous media, such as interstitial fluid and cytoplasm, are assumed to provide the dominant diffusion path. Here, the 'hydrophobic channeling' hypothesis is revisited, and several lines of evidence pointing toward lipid-accelerated oxygen diffusion pathways are discussed. The implications of hydrophobic channeling are considered in light of extended membrane networks in cells and tissues.

Reduced Oxygen Permeability upon Protein Incorporation Within Phospholipid Bilayers.

Intracellular oxygenation is key to energy metabolism as well as tumor radiation therapy. Although integral proteins are ubiquitous in membranes, few studies have considered their effects on molecular oxygen permeability. Published experimental work with rhodopsin and bacteriorhodopsin has led to the hypothesis that integral proteins lessen membrane oxygen permeability, as well as the permeability of the lipid region. The current work uses atomistic molecular dynamics simulations to test the influence of an ungated potassium channel protein on the oxygen permeability of palmitoyloleoylphosphatidylcholine (POPC) bilayers with and without cholesterol. Consistent with experiment, whole-membrane oxygen permeability is cut in half upon adding 30 wt% potassium channel protein to POPC, and the apparent permeability of the lipid portion of the membrane decreases by 40%. Unexpectedly, oxygen is found to interact directly with the protein surface, accompanied by a 40% reduction of the apparent whole-membrane diffusion coefficient. Similar effects are seen in systems combining the potassium channel with 1:1 POPC/cholesterol, but the magnitude of permeability reduction is smaller by ~30%. Overall, the simulations indicate that integral proteins can reduce oxygen permeability by altering the diffusional path and the local diffusivity. This effect may be especially important in the protein-dense membranes of mitochondria.

Predicted Decrease in Membrane Oxygen Permeability with Addition of Cholesterol.

Aberrations in cholesterol homeostasis are associated with several diseases that can be linked to changes in cellular oxygen usage. Prior biological and physical studies have suggested that membrane cholesterol content can modulate oxygen delivery, but questions of magnitude and biological significance remain open for further investigation. Here, we use molecular dynamics simulations in a first step toward reexamining the rate impact of cholesterol on the permeation of oxygen through phospholipid bilayers. The simulation models are closely compared with published electron paramagnetic resonance (EPR) oximetry measurements. The simulations predict an oxygen permeability reduction due to cholesterol but also suggest that the EPR experiments may have underestimated resistance to oxygen permeation in the phospholipid headgroup region.

Influence of Cholesterol on the Oxygen Permeability of Membranes: Insight from Atomistic Simulations.

Cholesterol is widely known to alter the physical properties and permeability of membranes. Several prior works have implicated cell membrane cholesterol as a barrier to tissue oxygenation, yet a good deal remains to be explained with regard to the mechanism and magnitude of the effect. We use molecular dynamics simulations to provide atomic-resolution insight into the influence of cholesterol on oxygen diffusion across and within the membrane. Our simulations show strong overall agreement with published experimental data, reproducing the shapes of experimental oximetry curves with high accuracy. We calculate the upper-limit transmembrane oxygen permeability of a 1-palmitoyl,2-oleoylphosphatidylcholine phospholipid bilayer to be 52 ± 2 cm/s, close to the permeability of a water layer of the same thickness. With addition of cholesterol, the permeability decreases somewhat, reaching 40 ± 2 cm/s at the near-saturating level of 62.5 mol % cholesterol and 10 ± 2 cm/s in a 100% cholesterol mimic of the experimentally observed noncrystalline cholesterol bilayer domain. These reductions in permeability can only be biologically consequential in contexts where the diffusional path of oxygen is not water dominated. In our simulations, cholesterol reduces the overall solubility of oxygen within the membrane but enhances the oxygen transport parameter (solubility-diffusion product) near the membrane center. Given relatively low barriers to passing from membrane to membrane, our findings support hydrophobic channeling within membranes as a means of cellular and tissue-level oxygen transport. In such a membrane-dominated diffusional scheme, the influence of cholesterol on oxygen permeability is large enough to warrant further attention.

Magnification of Cholesterol-Induced Membrane Resistance on the Tissue Level: Implications for Hypoxia.

High cellular membrane cholesterol is known to generate membrane resistance and reduce oxygen (O2) permeability. As such, cholesterol may contribute to the Warburg effect in tumor cells by stimulating intracellular hypoxia that cannot be detected from extracellular oxygen measurements. We probe the tissue-level impact of the phenomenon, asking whether layering of cells can magnify the influence of cholesterol, to modulate hypoxia in relation to capillary proximity. Using molecular dynamics simulations, we affirm that minimally hydrated, adjacent lipid bilayers have independent physical behavior. Combining this insight with published experimental data, we predict linearly increasing impact of membrane cholesterol on oxygen flux across cells layered in tissue.

Dimerization in the Grb7 protein.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.

Water-refined solution structure of the human Grb7-SH2 domain in complex with the erbB2 receptor peptide pY1139.

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.