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1.
Sci Adv ; 9(22): eadh4251, 2023 06 02.
Article in English | MEDLINE | ID: mdl-37256948

ABSTRACT

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.


Subject(s)
Ion Channels , Protons , Humans , Cryoelectron Microscopy , Ion Channels/chemistry , Mitochondrial Proteins/metabolism , Purine Nucleotides , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolism
2.
J Liposome Res ; 16(4): 373-89, 2006.
Article in English | MEDLINE | ID: mdl-17162579

ABSTRACT

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.


Subject(s)
Lipids/analysis , Lipids/chemistry , Peptides/administration & dosage , Peptides/chemistry , Transfection/instrumentation , Transfection/methods , Animals , Calorimetry , Cations/analysis , Cations/chemistry , Cell Survival/drug effects , Cells, Cultured , DNA/genetics , Fluoresceins , Genetic Vectors/genetics , Humans , Hydrogen-Ion Concentration , Lipids/toxicity , Liposomes , Male , Mice , Mice, Inbred C57BL , Molecular Structure
3.
Am J Pathol ; 165(5): 1663-76, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15509536

ABSTRACT

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.


Subject(s)
Dinoprostone/biosynthesis , Isoenzymes/physiology , Lung Injury , Prostaglandin-Endoperoxide Synthases/physiology , Animals , Bleomycin/pharmacology , Blotting, Western , Bronchoalveolar Lavage , Collagen/metabolism , Cyclooxygenase 2 , Dinoprostone/metabolism , Female , Fibroblasts/metabolism , Fibrosis/pathology , Heterozygote , Immunohistochemistry , Isoenzymes/genetics , Leukotrienes/metabolism , Lung/pathology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Time Factors , Up-Regulation
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