Biology subjects

Pohl, E. E.

Publications and source records attributed to Pohl, E. E..

7 recordsLinked to original sources

bioRxiv2025

Pro- and anti-inflammatory macrophages adjust UCP2 protein levels based on their intrinsic metabolism and available metabolites

The immune and metabolic responses of macrophages are closely linked, and mitochondria play a key role in polarizing them into pro-inflammatory (classical) and anti-inflammatory (alternative) states. Mitochondrial uncoupling protein 2 (UCP2) is involved in regulating macrophage inflammation and glucose metabolism; however, its regulatory mechanisms are unclear. We found that inflammatory stimuli reduce UCP2 expression and oxygen consumption rates (OCR), indicating mitochondrial suppression. Conversely, IL-4-activated macrophages displayed higher UCP2 levels and enhanced respiration. Under glucose deprivation, LPS-stimulated macrophages retained mitochondrial activity despite lower UCP2 levels. Pyruvate emerged as a key regulator of UCP2, blocking its mitochondrial entry reduced UCP2 expression. Additionally, hypoxia markedly decreased UCP2 levels in IL-4-activated macrophages, suggesting that hypoxia contributes to UCP2 suppression in pro-inflammatory macrophages. Notably, pro-inflammatory macrophages exhibit reduced reliance on UCP2 due to suppressed mitochondrial respiration. Pyruvate regulates UCP2 expression, highlighting the connection between glycolysis and mitochondrial metabolism. These findings may inform therapeutic strategies for diseases involving immune dysregulation.

biophysics↗

bioRxiv2025

The non-thermogenic function of opossum UCP1 is independent of its cytoplasmic binding network.

The primary function of uncoupling protein 1 (UCP1) is to mediate non-shivering thermogenesis in brown adipose tissue by facilitating proton transport across mitochondrial membranes. However, recent study has shown that UCP1 from the opossum (a marsupial) lacks thermogenic activity (Keipert et al., Science, 2024). This deficiency was attributed to two altered residues, K100 and Y289, within the cytosolic salt bridge network. Interestingly, the same residues are also present in UCP2 and UCP3 from murine species, both of which are known to transport protons in the presence of long-chain fatty acids (FAs). This raises questions about the validity of the proposed mechanism involving these residues in the loss of thermogenic function in opossum UCP1. In this study, we performed conductance measurements of planar lipid bilayers reconstituted with either recombinant mouse UCP1, or UCP1 mutant (Q100K/F289Y), or UCP2, or UCP3. Our data demonstrate that the conductance of the UCP1 double mutant, UCP2, and UCP3 is comparable to that of wild-type mouse UCP1 in the presence of palmitic or arachidonic acids. These findings suggest that the altered cytosolic residues (K100 and Y289) do not explain the lack of thermogenic function in opossum UCP1. Thus, the underlying molecular mechanism responsible for the absence of thermogenesis in opossum UCP1 remains unresolved, and further studies are warranted to elucidate the precise cause of this dysfunction. Given the emerging interest in UCP1 uncoupling as a potential therapeutic approach for treating obesity by increasing energy expenditure, understanding the molecular basis of UCP1s thermogenic dysfunction is of significant relevance.

biophysics↗

bioRxiv2023

The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of FA-activated proton transport.

Metabolic reprogramming in cancer cells has been linked to the mitochondrial dysfunction. Recent studies have suggested the mitochondrial 2-oxoglutarate/malate carrier (OGC) as a potential target for preventing cancer progression. Although OGC is known to be a part of the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. In this study, we investigated the contribution of recombinant murine OGC to the proton transport by measuring the conductance (Gm) of planar lipid bilayer membranes reconstituted with OGC. Our results show that OGC significantly increases Gm only in the presence of free fatty acids (FAs) and 2,4-dinitrophenol, demonstrating for the first time its involvement in proton transport. We found that (i) the increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of FAs, and (ii) OGC substrates and inhibitors compete with FAs for the same binding site. In addition, we have identified R90 as a crucial amino acid of the binding site for FAs, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. Elucidating the contribution of OGC to the uncoupling will be crucial in the design of targeted drugs for the treatment of cancer and other metabolic diseases.

biophysics↗

bioRxiv2022

Sex, stress and UCP2: mitochondria illuminate microglial identity beyond the dichotomy

Balanced and dynamic mitochondrial networks are essential for cell survival and function. Mitochondrial networks remodel their connectivity, content and subcellular localization to support optimized energy production under conditions of increased stress. In vivo, stressors can arise from the environment, such as in neuronal injury, or from mutation-induced cellular dysfunction. Cells programmed to identify and respond to these stress signals, like microglia, rely on optimized mitochondrial function, however we know very little about mitochondrial networks of microglia in vivo or their adaptation to environmental or cellular stressors. Here, we define the mitochondrial networks of retinal microglia in physiological conditions in vivo and evaluate network alterations by taking advantage of a microglia-selective mitochondria-labeled mouse model. First, we demonstrate significant differences in the mitochondrial networks of microglia in vivo and in vitro. Then, we induced neuronal injury in the in vivo environment using optic nerve crush, where responsive microglia exhibit more fragmented mitochondrial networks with increased content and perinuclear localization, supporting a state of increased cellular stress. Surprisingly, when we selectively increase cellular stress by knocking out the mitochondria-associated gene uncoupling protein 2 (UCP2), only male UCP2KO microglia establish a hyperfused mitochondrial network after injury, indicating sex differences in microglial stress mitigation. Ovariectomy in UCP2KO females elicits a shift toward the male hyperfused mitochondrial phenotype suggesting that circulating estrogens are a contributing factor to the differences in microglial stress mitigation.

neuroscience↗

bioRxiv2022

Membrane lipid reshaping underlies oxidative stress sensing by the mitochondrial proteins UCP1 and ANT1

Oxidative stress and ROS are important players in the pathogenesis of several diseases. Besides the direct modification of proteins, ROS modify lipids with negative spontaneous curvature, such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE-adducts potentiates the protonophoric activity of the uncoupling protein 1 (UCP1), but the molecular mechanism remains obscure. Here, we connected the ROS-mediated lipid shape alteration with the membrane mechanical properties and function of UCP1 and adenine nucleotide translocase 1 (ANT1). We found that lysophosphatidylcholines (OPC and MPC) and PE adducts decrease a bending modulus in lipid bilayers and increase the protonophoric activity of both proteins. Furthermore, MD simulations revealed that modified PEs and lysolipids alter the membrane lateral pressure profile in the same direction and range, indicating that modified PEs act as lipids with positive spontaneous curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. In conclusion, we demonstrate that UCP1 and ANT1 sense SCES and propose a new regulatory mechanism for the function of these proteins.

biophysics↗

bioRxiv2022

Mechanism of the ANT-mediated transport of fatty acid anions across the inner mitochondrial membrane

The additional protonophoric function of the mitochondrial adenine nucleotide translocase (ANT1) is now recognized. However, the molecular mechanism remains controversial. Fatty acid (FA) cycling hypothesis postulates that FAs transport protons across the inner mitochondrial membrane to the matrix by a flip-flop, whereas ANT1 facilitates the translocation of FA anions (FA-) back to the intermembrane space. By a combined approach involving measurements of current through the planar lipid bilayers reconstituted with recombinant ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that FA- is initially caught by R59 on the matrix side of ANT1, then moves along the positively charged protein-lipid interface, and binds to R79, where it is protonated in the hydrated cavity in the presence of D134. R79 is crucial for the competitive binding of ANT1 substrates (ATP and ADP) and inhibitors (carboxyatractyloside, bongkrekic acid). The binding sites are well-conserved in mitochondrial SLC25 members, implying a general transporting mechanism for FA anions.

biophysics↗

bioRxiv2020

Mitochondrial uncoupling protein 2 (UCP2), but not UCP3, is sensitive to oxygen concentration in cells

One of the important hallmarks of cardiovascular disease is mitochondrial dysfunction, which results in abnormal energy metabolism and increased ROS production in cardiomyocytes. Members of the mitochondrial uncoupling protein family, UCP2 and UCP3, are thought to be beneficial by reducing ROS due to mild uncoupling. More recent hypotheses suggest the involvement of both proteins in cell metabolism by the transport of yet unknown substrates. The protein expression pattern under physiological and pathological conditions is an important clue for the evaluation of UCP2/UCP3 function, however, there is still no consensus about it. Previously, we demonstrated that only UCP3 is present in the adult murine heart under physiological conditions and correlated it with the predominant use of fatty acids for oxidation. In contrast, UCP2 was found only in very young (stem cell – like) cardiomyocytes, that rely mostly on glycolysis. Here, we employed three different models (ex vivo heart ischemia-reperfusion model, myocardial infarction model, and embryonic stem cell differentiation into cardiomyocytes under hypoxic conditions) to evaluate the abundance of both proteins under ischemia and hypoxia conditions. We found that (i) oxygen shortage or bursts did not influence UCP3 levels in the heart and ii) UCP2 was not present in healthy, ischemic, or re-perfused hearts. However, (iii) UCP2 was sensitive to the oxygen concentration in stem cells, in which UCP2 is normally expressed. These results further support the idea, that two highly homologous proteins – UCP2 and UCP3 – are abundant in different cells and tissues, and differently regulated under physiological and pathological conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗