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.
Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism
Ferguson D, Eichler SJ, Yiew NKH, Colca JR, Cho K, Patti GJ, Shew TM, Lutkewitte AJ, Mukherjee S, McCommis KS, Niemi NM and Finck BN
Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism
Ferguson D, Eichler SJ, Yiew NKH, Colca JR, Cho K, Patti GJ, Shew TM, Lutkewitte AJ, Mukherjee S, McCommis KS, Niemi NM and Finck BN
The mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH.
Cryo-EM structure of coagulation factor V short
Mohammed BM, Pelc LA, Rau MJ and Di Cera E
Cryo-EM structure of coagulation factor V short
Mohammed BM, Pelc LA, Rau MJ and Di Cera E
Coagulation factor V (fV) is the inactive precursor of fVa, an essential component of the prothrombinase complex required for rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved due to intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and reveals the arrangement of the entire A1-A2-B-A3-C1-C2 assembly for the first time. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2 and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a likely binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S and fXa.
RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels
Clifford BL, Jarrett KE, Cheng J, Cheng A, Seldin M, Morand P, Lee R, Chen M, Baldan A, de Aguiar Vallim TQ and Tarling EJ
RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels
Clifford BL, Jarrett KE, Cheng J, Cheng A, Seldin M, Morand P, Lee R, Chen M, Baldan A, de Aguiar Vallim TQ and Tarling EJ
Removal of circulating plasma LDL-C (low-density lipoprotein cholesterol) by the liver relies on efficient endocytosis and intracellular vesicle trafficking. Increasing the availability of hepatic LDLRs (LDL receptors) remains a major clinical target for reducing LDL-C levels. Here, we describe a novel role for RNF130 (ring finger containing protein 130) in regulating plasma membrane availability of LDLR.
Monitoring prothrombin activation in plasma through loss of FRET
Stojanovski BM and Di Cera E
Monitoring prothrombin activation in plasma through loss of FRET
Stojanovski BM and Di Cera E
Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by substrate cleavage from other proteases. In addition, these assays depend on activation following cleavage at the R320 site of prothrombin and fail to report cleavage at the alternative R271 site that leads to shedding of the auxiliary Gla and kringle domains of prothrombin.
Comparison of Low-Density Lipoprotein Oxidation by Hydrophilic O( P)-Precursors and Lipid-O( P)-Precursor Conjugates
Maness PM, Cone GW, Ford DA and McCulla RD
Comparison of Low-Density Lipoprotein Oxidation by Hydrophilic O( P)-Precursors and Lipid-O( P)-Precursor Conjugates
Maness PM, Cone GW, Ford DA and McCulla RD
Lipid oxidation by reactive oxygen species (ROS) provide several different oxidation products that have been implicated in inflammatory responses. Ground state atomic oxygen [O( P)] is produced by the photodeoxygenation of certain heterocyclic oxides and has a reactivity that is unique from other ROS. Due to the reactive nature of O( P), the site of O( P)-generation is expected to influence the products in heterogenous solutions or environments. In this work, the oxidation of low-density lipoprotein (LDL) by lipids with covalently bound O( P)-photoprecursors was compared to more hydrophilic O( P)-photoprecursors. Lipid oxidation products were quantified after Bligh-Dyer extraction and pentafluorobenzyl bromide (PFB) derivatization by GC-MS. Unlike the more hydrophilic O( P)-photoprecursors, the oxidation of LDL during the irradiation of lipid-(O P)-photoprecursor conjugates showed little quenching by the addition of the O( P)-scavenging sodium allyl sulfonate. This indicated that lipophilic O( P)-photoprecursors are expected to generate lipid oxidation products where other more hydrophilic O( P)-photoprecursors could be quenched by other reactive groups present in solution or the environment.
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.
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.
Characterization of N-Acetyl Cysteine Adducts with Exogenous and Neutrophil-Derived 2-Chlorofatty Aldehyde
Shakya S, McGuffee RM and Ford DA
Characterization of N-Acetyl Cysteine Adducts with Exogenous and Neutrophil-Derived 2-Chlorofatty Aldehyde
Shakya S, McGuffee RM and Ford DA
Hypochlorous acid is produced by leukocyte myeloperoxidase activity. 2-Chlorofatty aldehydes (2-ClFALDs) are formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and are thus produced following leukocyte activation as well as in the lungs of mice exposed to chlorine gas. The biological role of 2-ClFALD is largely unknown. Recently, we used an alkyne analog (2-ClHDyA) of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDA), to identify proteins covalently modified by 2-ClHDyA in endothelial cells and epithelial cells. Here, we demonstrate that 2-ClHDA reduces the metabolic activity of RAW 264.7 cells in a dose-dependent manner. 2-ClHDyA localizes to the mitochondria, endoplasmic reticulum and Golgi in RAW 264.7 cells and modifies many proteins. The thiol-containing precursor of glutathione, N-acetyl cysteine (NAC), was shown to produce an adduct with 2-ClHDA with the loss of Cl (HDA-NAC). This adduct was characterized in both positive and negative ion modes using LC-MS/MS and electrospray ionization. NAC treatment of neutrophils reduced the 2-ClFALD levels in PMA-stimulated cells with subsequent increases in HDA-NAC. NAC treatments reduced the 2-ClHDA-elicited loss of metabolic activity in RAW 264.7 cells as well as 2-ClHDA protein modification. These studies demonstrate that 2-ClFALD toxic effects can be reduced by NAC, which reduces protein modification.
The Hepatic Mitochondrial Pyruvate Carrier as a Regulator of Systemic Metabolism and a Therapeutic Target for Treating Metabolic Disease
McCommis KS and Finck BN
The Hepatic Mitochondrial Pyruvate Carrier as a Regulator of Systemic Metabolism and a Therapeutic Target for Treating Metabolic Disease
McCommis KS and Finck BN
Pyruvate sits at an important metabolic crossroads of intermediary metabolism. As a product of glycolysis in the cytosol, it must be transported into the mitochondrial matrix for the energy stored in this nutrient to be fully harnessed to generate ATP or to become the building block of new biomolecules. Given the requirement for mitochondrial import, it is not surprising that the mitochondrial pyruvate carrier (MPC) has emerged as a target for therapeutic intervention in a variety of diseases characterized by altered mitochondrial and intermediary metabolism. In this review, we focus on the role of the MPC and related metabolic pathways in the liver in regulating hepatic and systemic energy metabolism and summarize the current state of targeting this pathway to treat diseases of the liver. Available evidence suggests that inhibiting the MPC in hepatocytes and other cells of the liver produces a variety of beneficial effects for treating type 2 diabetes and nonalcoholic steatohepatitis. We also highlight areas where our understanding is incomplete regarding the pleiotropic effects of MPC inhibition.