Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature
Coll-Bonfill N, Mahajan U, Shashkova EV, Lin CJ, Mecham RP and Gonzalo S
Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature
Coll-Bonfill N, Mahajan U, Shashkova EV, Lin CJ, Mecham RP and Gonzalo S
Hutchinson-Gilford progeria syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated prelamin A protein "progerin" that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially detrimental for vascular smooth muscle cells (VSMC). Vessel stiffening and aortic atherosclerosis in HGPS patients are accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here, we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNA-seq analysis shows induction of a profibrotic and pro-inflammatory aging-associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and pro-inflammatory signatures contribute to vascular stiffness in HGPS.
Myeloperoxidase-derived hypochlorous acid targets human airway epithelial plasmalogens liberating protein modifying electrophilic 2-chlorofatty aldehydes
Shakya S, Pyles KD, Albert CJ, Patel RP, McCommis KS and Ford DA
Myeloperoxidase-derived hypochlorous acid targets human airway epithelial plasmalogens liberating protein modifying electrophilic 2-chlorofatty aldehydes
Shakya S, Pyles KD, Albert CJ, Patel RP, McCommis KS and Ford DA
Neutrophil and airway epithelial cell interactions are critical in the inflammatory response to viral infections including respiratory syncytial virus, Sendai virus, and SARS-CoV-2. Airway epithelial cell dysfunction during viral infections is likely mediated by the interaction of virus and recruited neutrophils at the airway epithelial barrier. Neutrophils are key early responders to viral infection. Neutrophil myeloperoxidase catalyzes the conversion of hydrogen peroxide to hypochlorous acid (HOCl). Previous studies have shown HOCl targets host neutrophil and endothelial cell plasmalogen lipids, resulting in the production of the chlorinated lipid, 2-chlorofatty aldehyde (2-ClFALD). We have previously shown that the oxidation product of 2-ClFALD, 2-chlorofatty acid (2-ClFA) is present in bronchoalveolar lavage fluid of Sendai virus-infected mice, which likely results from the attack of the epithelial plasmalogen by neutrophil-derived HOCl. Herein, we demonstrate small airway epithelial cells contain plasmalogens enriched with oleic acid at the sn-2 position unlike endothelial cells which contain arachidonic acid enrichment at the sn-2 position of plasmalogen. We also show neutrophil-derived HOCl targets epithelial cell plasmalogens to produce 2-ClFALD. Further, proteomics and over-representation analysis using the ω-alkyne analog of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDyA) showed cell adhesion molecule binding and cell-cell junction enriched categories similar to that observed previously in endothelial cells. However, in contrast to endothelial cells, proteins in distinct metabolic pathways were enriched with 2-ClFALD modification, particularly pyruvate metabolism was enriched in epithelial cells and mitochondrial pyruvate respiration was reduced. Collectively, these studies demonstrate, for the first time, a novel plasmalogen molecular species distribution in airway epithelial cells that are targeted by myeloperoxidase-derived hypochlorous acid resulting in electrophilic 2-ClFALD, which potentially modifies epithelial physiology by modifying proteins.
Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors
Short KM, Estiarte MA, Pham SM, Williams DC, Igoudin L, Dash S, Sandoval N, Datta A, Pozzi N, Di Cera E and Kita DB
Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors
Short KM, Estiarte MA, Pham SM, Williams DC, Igoudin L, Dash S, Sandoval N, Datta A, Pozzi N, Di Cera E and Kita DB
Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.
Mitochondrial carbonic anhydrase VA and VB: properties and roles in health and disease
Aspatwar A, Supuran CT, Waheed A, Sly WS and Parkkila S
Mitochondrial carbonic anhydrase VA and VB: properties and roles in health and disease
Aspatwar A, Supuran CT, Waheed A, Sly WS and Parkkila S
Carbonic anhydrase V (CA V), a mitochondrial enzyme, was first isolated from guinea-pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CA VA enzyme is most highly expressed in the liver, whereas CA VB shows a broad tissue distribution. Car5A knockout mice demonstrated a predominant role for CA VA in ammonia detoxification, whereas the roles of CA VB in ureagenesis and gluconeogenesis were evident only in the absence of CA VA. Previous studies have suggested that CA VA is mainly involved in the provision of HCO for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolic profiles of these children showed a reduced supply of HCO to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. Although the role of CA VB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells, which sustain spermatogenesis. Metabolic disease associated with CA VA appears to be more common than other inborn errors of metabolism and responds well to treatment with N-carbamyl-l-glutamate. Therefore, early identification of hyperammonaemia will allow specific treatment with N-carbamyl-l-glutamate and prevent neurological sequelae. Carbonic anhydrase VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonaemia in neonates and young children.
Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit
Elliff J, Biswas A, Roshan P, Kuppa S, Patterson A, Mattice J, Chinnaraj M, Burd R, Walker SE, Pozzi N, Antony E, Bothner B and Origanti S
Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit
Elliff J, Biswas A, Roshan P, Kuppa S, Patterson A, Mattice J, Chinnaraj M, Burd R, Walker SE, Pozzi N, Antony E, Bothner B and Origanti S
Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.
Comparative sequence analysis of vitamin K-dependent coagulation factors
Stojanovski BM and Di Cera E
Comparative sequence analysis of vitamin K-dependent coagulation factors
Stojanovski BM and Di Cera E
Prothrombin, protein C, and factors VII, IX, and X are vitamin K (VK)-dependent coagulation proteins that play an important role in the initiation, amplification, and subsequent attenuation of the coagulation response. Blood coagulation evolved in the common vertebrate ancestor as a specialization of the complement system and immune response, which in turn bear close evolutionary ties with developmental enzyme cascades. There is currently no comprehensive analysis of the evolutionary changes experienced by these coagulation proteins during the radiation of vertebrates and little is known about conservation of residues that are important for zymogen activation and catalysis.
Structural Basis of Sequential and Concerted Cooperativity
Morea V, Angelucci F, Tame JRH, Di Cera E and Bellelli A
Structural Basis of Sequential and Concerted Cooperativity
Morea V, Angelucci F, Tame JRH, Di Cera E and Bellelli A
Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the "concerted model" by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the "sequential model" originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms.
Cryo-EM structures of coagulation factors
Di Cera E, Mohammed BM, Pelc LA and Stojanovski BM
Cryo-EM structures of coagulation factors
Di Cera E, Mohammed BM, Pelc LA and Stojanovski BM
A State of the Art lecture titled "Cryo-EM structures of coagulation factors" was presented at the ISTH Congress in 2022. Cryogenic electron microscopy (cryo-EM) is a revolutionary technique capable of solving the structure of high molecular weight proteins and their complexes, unlike nuclear magnetic resonance (NMR), and under conditions not biased by crystal contacts, unlike X-ray crystallography. These features are particularly relevant to the analysis of coagulation factors that are too big for NMR and often recalcitrant to X-ray investigation. Using cryo-EM, we have solved the structures of coagulation factors V and Va, prothrombinase on nanodiscs, and the prothrombin-prothrombinase complex. These structures have advanced basic knowledge in the field of thrombosis and hemostasis, especially on the function of factor V and the molecular mechanism for prothrombin activation, and set the stage for exciting new lines of investigation. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.
Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking
Palladino END, Bernas T, Green CD, Weigel C, Singh SK, Senkal CE, Martello A, Kennelly JP, Bieberich E, Tontonoz P, Ford DA, Milstien S, Eden ER and Spiegel S
Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking
Palladino END, Bernas T, Green CD, Weigel C, Singh SK, Senkal CE, Martello A, Kennelly JP, Bieberich E, Tontonoz P, Ford DA, Milstien S, Eden ER and Spiegel S
Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.
strain-dependent lipid alterations in cocultures with endothelial cells and neutrophils modeling sepsis
Amunugama K, Pike DP and Ford DA
strain-dependent lipid alterations in cocultures with endothelial cells and neutrophils modeling sepsis
Amunugama K, Pike DP and Ford DA
Dysregulated lipid metabolism is common in infection and inflammation and is a part of the complex milieu underlying the pathophysiological sequelae of disease. Sepsis is a major cause of mortality and morbidity in the world and is characterized by an exaggerated host response to an infection. Metabolic changes, including alterations in lipid metabolism, likely are important in sepsis pathophysiology. Here, we designed an cell culture model using endothelial cells, , and neutrophils to mimic sepsis in a simplified cell model. Lipid alterations were studied in the presence of the pathogenic strain CFT073 and non-pathogenic strain JM109. We employed untargeted lipidomics to first identify lipid changes and then targeted lipidomics to confirm changes. Both unique and shared lipid signatures were identified in cocultures with these strains. In the absence of neutrophils, the CFT073 strain elicited alterations in lysophosphatidylcholine and diglyceride molecular species during coculture while both strains led to increases in phosphatidylglycerols. Lipid alterations in these cocultures changed with the addition of neutrophils. In the presence of neutrophils with and endothelial cells, triglyceride increases were a unique response to the CFT073 strain while phosphatidylglycerol and diglyceride increases occurred in response to both strains. Phosphatidylethanolamine also increased in neutrophils, and endothelial cells cocultures, and this response was greater in the presence of the CFT073 strain. We further evaluated changes in phosphatidylethanolamine in a rat model of sepsis, which showed multiple plasma phosphatidylethanolamine molecular species were elevated shortly after the induction of sepsis. Collectively, these findings demonstrate unique lipid responses by co-cultures of with endothelial cells which are dependent on the strain as well as the presence of neutrophils. Furthermore, increases in phosphatidylethanolamine levels in CFT073 urosepsis , endothelial cell, neutrophil cocultures were similarly observed in the plasma of septic rats.