Structural determinants of PAR1 cleavage by activated protein C
Stojanovski BM and Di Cera E
Structural determinants of PAR1 cleavage by activated protein C
Stojanovski BM and Di Cera E
Activated protein C (APC) performs cytoprotective functions mediated by cleavage of the protease activated receptor 1 (PAR1) in the presence of the endothelial protein C receptor (EPCR) and signaling through b-arrestin-2. APC cleaves PAR1 at R41 and R46, but the specificity of the reaction is low. In contrast, thrombin cleaves PAR1 at R41 only in a reaction that is independent of EPCR, produces a pro-inflammatory response mediated by signaling through G-protein intermediates and features high specificity. The molecular basis of this difference between APC and thrombin remains unknown.
At last: the mitochondrial pyruvate carrier structure revealed!
Finck BN, Hadfield CM and McCommis KS
At last: the mitochondrial pyruvate carrier structure revealed!
Finck BN, Hadfield CM and McCommis KS
Mitochondrial pyruvate carrier (MPC) inhibitors have shown promise as therapeutics for treating several chronic diseases. However, the structure of MPC and the molecular mechanisms by which it interacts with inhibitors have remained unclear, impeding rational drug design. Multiple groups have now independently resolved the structure of the MPC heterodimer.
Human lung microvascular endothelial cell protein modification by 2-chlorohexadecanoic acid: RhoA mediates 2-chlorohexadecanoic acid-elicited endothelial activation
Carlson HL and Ford DA
Human lung microvascular endothelial cell protein modification by 2-chlorohexadecanoic acid: RhoA mediates 2-chlorohexadecanoic acid-elicited endothelial activation
Carlson HL and Ford DA
Chlorolipids are produced during the neutrophil respiratory burst as a result of myeloperoxidase (MPO)-generated hypochlorous acid (HOCl) targeting the vinyl ether bond of plasmalogen phospholipids. The initial products of this reaction are 2-chlorofatty aldehydes (2-ClFALDs), which are subsequently oxidized to 2-chlorofatty acids (2-ClFAs). 2-Chlorohexadecanoic acid (2-ClHA) is the 16-carbon 2-ClFA species, and previous studies have shown that increased levels of plasma 2-ClHA associate with acute respiratory distress syndrome (ARDS)-caused mortality in human sepsis. 2-ClHA causes endothelial barrier dysfunction and increases neutrophil and platelet adherence to the endothelium. In this study, click chemistry analogs of 2-ClHA and hexadecanoic acid (HA) were used to identify proteins covalently modified by 2-ClHA and HA in human lung microvascular endothelial cells (HLMVECs). Eleven proteins were specifically modified by 2-ClHA, and an additional one hundred and ninety-four proteins were modified by both 2-ClHA and HA. STRING analysis of 2-ClHA-modified proteins revealed a network of proteins with RhoA as a hub. RhoA is one of the proteins specifically modified by 2-ClHA and not HA. The RhoA inhibitors, Rhosin and C3, inhibited both 2-ClHA-elicited HLMVEC barrier dysfunction and angiopoietin-2 (Ang-2) release from HLMVEC. Further studies showed 2-ClHA activates HLMVEC RhoA activity. The specificity of the 2-ClHA-RhoA pathway for endothelial activation was further confirmed since HA did not cause HLMVEC barrier dysfunction, Ang-2 release and RhoA activation. Collectively, these studies have identified multiple proteins modified exclusively by 2-ClHA in HLMVECs, including RhoA. These proteomics studies led to the key finding that RhoA is an important mediator of 2-ClHA-caused endothelial barrier dysfunction.
Structural dynamics of sphingosine kinase 1 regulation and inhibition
Abd Emami B, Shubbar A, Woods H, Moradi M and Dastvan R
Structural dynamics of sphingosine kinase 1 regulation and inhibition
Abd Emami B, Shubbar A, Woods H, Moradi M and Dastvan R
Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate, a bioactive lipid implicated in cancer progression and other diseases. Despite its clinical relevance, the structural and dynamic basis of SK1 regulation and inhibition remains poorly understood. Using an integrated spectroscopic and computational approach, we uncover conformational transitions that govern substrate entry, catalysis, and inhibitor binding. Phosphorylation of Ser225 triggers regulatory loop rearrangements and salt bridge reshuffling, priming SK1 for membrane engagement and catalytic activity. We identify a previously uncharacterized catalytic intermediate featuring a distinct conformation with a highly dynamic lipid-binding loop 1 (LBL-1), sensitive to potent inhibitors such as PF-543. This inhibitor locks SK1 in an inactive state by restricting LBL-1 dynamics and globally stabilizing a non-catalytic conformation. Notably, SK1 forms functionally distinct dimers stabilized by ligand or membrane interactions, revealing a dynamic, multilayered regulatory mechanism governed by structural flexibility. These findings define a novel inhibitory mechanism and offer a structural framework for developing next-generation SK1-targeted therapeutics.
Technical recommendations for analyzing oxylipins by liquid chromatography-mass spectrometry
Schebb NH, Kampschulte N, Hagn G, Plitzko K, Meckelmann SW, Ghosh S, Joshi R, Kuligowski J, Vuckovic D, Botana MT, Sánchez-Illana Á, Zandkarimi F, Das A, Yang J, Schmidt L, Checa A, Roche HM, Armando AM, Edin ML, Lih FB, Aristizabal-Henao JJ, Miyamoto S, Giuffrida F, Moussaieff A, Domingues R, Rothe M, Hinz C, Das US, Rund KM, Taha AY, Hofstetter RK, Werner M, Werz O, Kahnt AS, Bertrand-Michel J, Le Faouder P, Gurke R, Thomas D, Torta F, Milic I, Dias IHK, Spickett CM, Biagini D, Lomonaco T, Idborg H, Liu JY, Fedorova M, Ford DA, Barden A, Mori TA, Kennedy PD, Maxey K, Ivanisevic J, Gallart-Ayala H, Gladine C, Wenk M, Galano JM, Durand T, Stark KD, Barbas C, Garscha U, Gelhaus SL, Ceglarek U, Flamand N, Griffin JL, Ahrends R, Arita M, Zeldin DC, Schopfer FJ, Quehenberger O, Julian R, Nicolaou A, Blair IA, Murphy MP, Hammock BD, Freeman B, Liebisch G, Serhan CN, Köfeler HC, Jakobsson PJ, Steinhilber D, Gelb MH, Holčapek M, Andrew R, Giera M, FitzGerald GA, Murphy RC, Newman JW, Dennis EA, Ekroos K, Milne GL, Gijón MA, Vesper HW, Wheelock CE and O'Donnell VB
Technical recommendations for analyzing oxylipins by liquid chromatography-mass spectrometry
Schebb NH, Kampschulte N, Hagn G, Plitzko K, Meckelmann SW, Ghosh S, Joshi R, Kuligowski J, Vuckovic D, Botana MT, Sánchez-Illana Á, Zandkarimi F, Das A, Yang J, Schmidt L, Checa A, Roche HM, Armando AM, Edin ML, Lih FB, Aristizabal-Henao JJ, Miyamoto S, Giuffrida F, Moussaieff A, Domingues R, Rothe M, Hinz C, Das US, Rund KM, Taha AY, Hofstetter RK, Werner M, Werz O, Kahnt AS, Bertrand-Michel J, Le Faouder P, Gurke R, Thomas D, Torta F, Milic I, Dias IHK, Spickett CM, Biagini D, Lomonaco T, Idborg H, Liu JY, Fedorova M, Ford DA, Barden A, Mori TA, Kennedy PD, Maxey K, Ivanisevic J, Gallart-Ayala H, Gladine C, Wenk M, Galano JM, Durand T, Stark KD, Barbas C, Garscha U, Gelhaus SL, Ceglarek U, Flamand N, Griffin JL, Ahrends R, Arita M, Zeldin DC, Schopfer FJ, Quehenberger O, Julian R, Nicolaou A, Blair IA, Murphy MP, Hammock BD, Freeman B, Liebisch G, Serhan CN, Köfeler HC, Jakobsson PJ, Steinhilber D, Gelb MH, Holčapek M, Andrew R, Giera M, FitzGerald GA, Murphy RC, Newman JW, Dennis EA, Ekroos K, Milne GL, Gijón MA, Vesper HW, Wheelock CE and O'Donnell VB
Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenges. As members of the lipidomics community, we developed technical recommendations to ensure best practices when quantifying oxylipins by LC-MS.
Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates
Yan X, Kuster D, Mohanty P, Nijssen J, Pombo-García K, Garcia Morato J, Rizuan A, Franzmann TM, Sergeeva A, Ly AM, Liu F, Passos PM, George L, Wang SH, Shenoy J, Danielson HL, Ozguney B, Honigmann A, Ayala YM, Fawzi NL, Dickson DW, Rossoll W, Mittal J, Alberti S and Hyman AA
Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates
Yan X, Kuster D, Mohanty P, Nijssen J, Pombo-García K, Garcia Morato J, Rizuan A, Franzmann TM, Sergeeva A, Ly AM, Liu F, Passos PM, George L, Wang SH, Shenoy J, Danielson HL, Ozguney B, Honigmann A, Ayala YM, Fawzi NL, Dickson DW, Rossoll W, Mittal J, Alberti S and Hyman AA
Cytosolic aggregation of the nuclear protein TAR DNA-binding protein 43 (TDP-43) is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43-enriched phase within stress granules, which subsequently transition into pathological aggregates. Intra-condensate demixing of TDP-43 is observed in iPS-motor neurons, a disease mouse model, and patient samples. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We suggest that up-concentration inside condensates followed by intra-condensate demixing could be a general pathway for protein aggregation.
Lupus anticoagulant and antiprothrombin antibodies: embracing the future
Pengo V and Pozzi N
Lupus anticoagulant and antiprothrombin antibodies: embracing the future
Pengo V and Pozzi N
Lupus anticoagulant (LAC) is a well-known laboratory test used to explore potential reasons for the prolongation of phospholipid-dependent coagulation tests. An extended clotting time in a coagulation test typically suggests a bleeding tendency, as the plasma takes longer to clot. However, a positive LAC result, defined as normalization of prolonged clotting time by adding anionic phospholipids in the system, does not necessarily imply this. In fact, quite the opposite is true: a positive LAC often strongly correlates with an increased risk of thromboembolic events. Therefore, despite being conceptually counterintuitive, LAC remains extremely valuable in routine clinical practice for identifying individuals at risk for thromboembolic events. Over the years, various factors have been recognized as potential inducers of LAC, with antiphospholipid antibodies associated with antiphospholipid syndrome (APS) playing a significant role. Today, research indicates that, among antiphospholipid antibodies, those targeting plasma proteins β-glycoprotein I and prothrombin are central to LAC. This article offers a historical perspective on LAC, emphasizing recent developments in antiprothrombin antibodies, their connection to LAC, and novel detection methods. Our premise is that a deeper understanding of how antiprothrombin antibodies contribute to LAC and the identification of subpopulations of these antibodies potentially responsible for it in thrombotic APS patients could lead to transformative advancements, offering new strategies for risk stratification and personalized treatments for patients with APS and beyond.
Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR
Kashyap R, Walsh N, Deveryshetty J, Tokmina-Lukaszewska M, Zhao K, Gan YJ, Hoffman BM, Sarangi R, Bothner B, Bennett B and Antony E
Cryo-EM captures the coordination of asymmetric electron transfer through a di-copper site in DPOR
Kashyap R, Walsh N, Deveryshetty J, Tokmina-Lukaszewska M, Zhao K, Gan YJ, Hoffman BM, Sarangi R, Bothner B, Bennett B and Antony E
Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an αβ heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions.
Cryo-EM structure of coagulation factor Va bound to activated protein C
Mohammed BM, Basore K and Di Cera E
Cryo-EM structure of coagulation factor Va bound to activated protein C
Mohammed BM, Basore K and Di Cera E
Coagulation factor Va (fVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, fVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the fVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of fVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here we report the cryo-EM structure of the fVa-APC complex at 3.15 Å resolution where the protease domain of APC engages R506 in the A2 domain of fVa mainly through electrostatic interactions between positively charged residues in the 30- and 70- loops of APC and an electronegative surface of fVa. The auxiliary Gla and EGF domains of APC are highly dynamic and point to solvent, without making contacts with fVa. Binding of APC displaces a large portion of the A2 domain of fVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch", or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the fVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.
2-Chloro- and 2-Bromopalmitic acids inhibit mitochondrial function in airway epithelial cells
Ricart K, McCommis KS, Ford DA and Patel RP
2-Chloro- and 2-Bromopalmitic acids inhibit mitochondrial function in airway epithelial cells
Ricart K, McCommis KS, Ford DA and Patel RP
2-Chloropalmitic acid (2-ClPA) and 2-bromopalmitic acid (2-BrPa) increase in inflammatory lung disease associated with formation of hypochlorous or hypobromous acid, and exposure to halogen gases. Moreover, these lipids may elicit cell responses that contribute to lung injury, but the mechanisms remain unclear. Here, we tested the hypothesis that 2-ClPA and 2-BrPA induce metabolic defects in airway epithelial cells by targeting mitochondria. H441 or primary human airway epithelial cells were treated with 2-ClPA or 2-BrPA and bioenergetics measured using oxygen consumption rates and extracellular acidification rates, as well as respiratory complex activities. Relative to vehicle or palmitic acid, both 2-halofatty acids inhibited ATP-linked oxygen consumption and reserve capacity, suggestive of increased proton leak. However, neither 2-ClPA nor 2-BrPA altered mitochondrial membrane potential, suggesting proton leak does not underlie inhibited ATP-linked oxygen consumption. Interestingly, complex II activity was significantly inhibited which may contribute to diminished reserve capacity, but activity of complexes I, III and IV remain unchanged. Taken together, the presented data highlight the potential of 2-halofatty acids to disrupt bioenergetics and in turn cause cellular dysfunction.