Probing the conformational dynamics of thiol-isomerases using non-canonical amino acids and single-molecule FRET
Ponzar N and Pozzi N
Probing the conformational dynamics of thiol-isomerases using non-canonical amino acids and single-molecule FRET
Ponzar N and Pozzi N
Disulfide bonds drive protein correct folding, prevent protein aggregation, and stabilize three-dimensional structures of proteins and their assemblies. Dysregulation of this activity leads to several disorders, including cancer, neurodegeneration, and thrombosis. A family of 20+ enzymes, called thiol-isomerases (TIs), oversee this process in the endoplasmic reticulum of human cells to ensure efficacy and accuracy. While the biophysical and biochemical properties of cysteine residues are well-defined, our structural knowledge of how TIs select, interact and process their substrates remains poorly understood. How TIs structurally and functionally respond to changes in redox environment and other post-translational modifications remain unclear, too. We recently developed a workflow for site-specific incorporation of non-canonical amino acids into protein disulfide isomerase (PDI), the prototypical member of TIs. Combined with click chemistry, this strategy enabled us to perform single-molecule biophysical studies of PDI under various solution conditions. This paper details protocols and discusses challenges in performing these experiments. We expect this approach, combined with other emerging technologies in single-molecule biophysics and structural biology, to facilitate the exploration of the mechanisms by which TIs carry out their fascinating but poorly understood roles in humans, especially in the context of thrombosis.
Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics
Hoitsma NM, Norris J, Khoang TH, Kaushik V, Chadda R, Antony E, Hedglin M and Freudenthal BD
Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics
Hoitsma NM, Norris J, Khoang TH, Kaushik V, Chadda R, Antony E, Hedglin M and Freudenthal BD
Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.
Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Reveals Remarkable Role of Fluorine
Schubert TJ, Oboh E, Peek H, Philo E, Teixeira JE, Stebbins EE, Miller P, Oliva J, Sverdrup FM, Griggs DW, Huston CD and Meyers MJ
Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Reveals Remarkable Role of Fluorine
Schubert TJ, Oboh E, Peek H, Philo E, Teixeira JE, Stebbins EE, Miller P, Oliva J, Sverdrup FM, Griggs DW, Huston CD and Meyers MJ
Our previous work identified compound (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite (EC = 0.17 μM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (, EC = 0.07 μΜ), which was found to be orally efficacious with an ED < 5 mg/kg BID in a -infection mouse model, superior to SLU-2633.
An Aurora B-RPA signaling axis secures chromosome segregation fidelity
Roshan P, Kuppa S, Mattice JR, Kaushik V, Chadda R, Pokhrel N, Tumala BR, Biswas A, Bothner B, Antony E and Origanti S
An Aurora B-RPA signaling axis secures chromosome segregation fidelity
Roshan P, Kuppa S, Mattice JR, Kaushik V, Chadda R, Pokhrel N, Tumala BR, Biswas A, Bothner B, Antony E and Origanti S
Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.
Membrane phospholipid remodeling modulates nonalcoholic steatohepatitis progression by regulating mitochondrial homeostasis
Tian Y, Jellinek MJ, Mehta K, Seok SM, Kuo SH, Lu W, Shi R, Lee R, Lau GW, Kemper JK, Zhang K, Ford DA and Wang B
Membrane phospholipid remodeling modulates nonalcoholic steatohepatitis progression by regulating mitochondrial homeostasis
Tian Y, Jellinek MJ, Mehta K, Seok SM, Kuo SH, Lu W, Shi R, Lee R, Lau GW, Kemper JK, Zhang K, Ford DA and Wang B
NASH, characterized by inflammation and fibrosis, is emerging as a leading etiology of HCC. Lipidomics analyses in the liver have shown that the levels of polyunsaturated phosphatidylcholine (PC) are decreased in patients with NASH, but the roles of membrane PC composition in the pathogenesis of NASH have not been investigated. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), a phospholipid (PL) remodeling enzyme that produces polyunsaturated PLs, is a major determinant of membrane PC content in the liver.
Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation
Yang M, Chiu J, Scartelli C, Ponzar N, Patel S, Patel A, Ferreira RB, Keyes RF, Carroll KS, Pozzi N, Hogg PJ, Smith BC and Flaumenhaft R
Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation
Yang M, Chiu J, Scartelli C, Ponzar N, Patel S, Patel A, Ferreira RB, Keyes RF, Carroll KS, Pozzi N, Hogg PJ, Smith BC and Flaumenhaft R
Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines.
Hepatic Phospholipid Remodeling Modulates Insulin Sensitivity and Systemic Metabolism
Tian Y, Mehta K, Jellinek MJ, Sun H, Lu W, Shi R, Ingram K, Friedline RH, Kim JK, Kemper JK, Ford DA, Zhang K and Wang B
Hepatic Phospholipid Remodeling Modulates Insulin Sensitivity and Systemic Metabolism
Tian Y, Mehta K, Jellinek MJ, Sun H, Lu W, Shi R, Ingram K, Friedline RH, Kim JK, Kemper JK, Ford DA, Zhang K and Wang B
The liver plays a central role in regulating glucose and lipid metabolism. Aberrant insulin action in the liver is a major driver of selective insulin resistance, in which insulin fails to suppress glucose production but continues to activate lipogenesis in the liver, resulting in hyperglycemia and hypertriglyceridemia. The underlying mechanisms of selective insulin resistance are not fully understood. Here It is shown that hepatic membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) regulates insulin signaling and systemic glucose and lipid metabolism. Hyperinsulinemia induced by high-fat diet (HFD) feeding augments hepatic Lpcat3 expression and membrane unsaturation. Loss of Lpcat3 in the liver improves insulin resistance and blunts lipogenesis in both HFD-fed and genetic ob/ob mouse models. Mechanistically, Lpcat3 deficiency directly facilitates insulin receptor endocytosis, signal transduction, and hepatic glucose production suppression and indirectly enhances fibroblast growth factor 21 (FGF21) secretion, energy expenditure, and glucose uptake in adipose tissue. These findings identify hepatic LPCAT3 and membrane phospholipid composition as a novel regulator of insulin sensitivity and provide insights into the pathogenesis of selective insulin resistance.
Monitoring prothrombin activation in plasma through loss of Förster resonance energy transfer
Stojanovski BM and Di Cera E
Monitoring prothrombin activation in plasma through loss of Förster resonance energy transfer
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 prothrombin R320 site and fail to report the cleavage at the alternative R271 site, leading to the 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 PF, 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 PF, 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.
Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis
Sharpe MC, Pyles KD, Hallcox T, Kamm DR, Piechowski M, Fisk B, Albert CJ, Carpenter DH, Ulmasov B, Ford DA, Neuschwander-Tetri BA and McCommis KS
Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis
Sharpe MC, Pyles KD, Hallcox T, Kamm DR, Piechowski M, Fisk B, Albert CJ, Carpenter DH, Ulmasov B, Ford DA, Neuschwander-Tetri BA and McCommis KS
Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH.