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Biotechnology and Bioengineering Jun 2024The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant...
The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).
PubMed: 38874319
DOI: 10.1002/bit.28766 -
Nature Communications Jun 2024Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well...
Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.
Topics: Talin; Focal Adhesions; Cell Membrane; Vinculin; Humans; Animals; Phosphatidylinositol 4,5-Diphosphate; Integrins; Cytoplasm; Protein Binding; Phase Separation
PubMed: 38862544
DOI: 10.1038/s41467-024-49222-z -
Plant Physiology and Biochemistry : PPB Jun 2024The enzyme phospholipase A (PLA) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in...
The enzyme phospholipase A (PLA) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A (MiPLA) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA contained 3 introns. Mature MiPLA (mMiPLA) had a conserved Ca-binding loop and a catalytic site motif that has been recognized in plant secretory PLA (sPLA) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA was clustered within GroupXIA of plant sPLA proteins. To ascertain the function of MiPLA, the cDNA coding for mMiPLA was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA in Escherichia coli. Recombinant mMiPLA was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA. Although recombinant mMiPLA displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA illustrated that MiPLA was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA transcription and free ArA levels were established. Consequently, the role of mMiPLA in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.
PubMed: 38861822
DOI: 10.1016/j.plaphy.2024.108806 -
Journal of Biochemistry Jun 2024Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate...
Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids, and generated CSA- and CSE-liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE-liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS-liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.
PubMed: 38861406
DOI: 10.1093/jb/mvae041 -
Analytical Chemistry Jun 2024Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions...
Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.
Topics: Cardiolipins; Mass Spectrometry; Mutation; Aquaporins; Binding Sites; Protein Binding; Membrane Proteins; Thermodynamics; Models, Molecular; Escherichia coli Proteins
PubMed: 38859611
DOI: 10.1021/acs.analchem.4c01704 -
BMC Genomics Jun 2024Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in...
BACKGROUND
Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in plant development and stress responses is well documented.
RESULTS
In the present investigation, a thorough genome-wide analysis revealed that the pearl millet genome contains at least 44 phospholipase genes distributed across its 7 chromosomes, with chromosome one harbouring the highest number of these genes. The synteny analysis suggested a close genetic relationship of pearl millet phospholipases with that of foxtail millet and sorghum. All identified genes were examined to unravel their gene structures, protein attributes, cis-regulatory elements, and expression patterns in two pearl millet genotypes contrasting for rancidity. All the phospholipases have a high alpha-helix content and distorted regions within the predicted secondary structures. Moreover, many of these enzymes possess binding sites for both metal and non-metal ligands. Additionally, the putative promoter regions associated with these genes exhibit multiple copies of cis-elements specifically responsive to biotic and abiotic stress factors and signaling molecules. The transcriptional profiling of 44 phospholipase genes in two genotypes contrasting for rancidity across six key tissues during pearl millet growth revealed a predominant expression in grains, followed by seed coat and endosperm. Specifically, the genes PgPLD-alpha1-1, PgPLD-alpha1-5, PgPLD-delta1-7a, PgPLA1-II-1a, and PgPLD-delta1-2a exhibited notable expression in grains of both the genotypes while showing negligible expression in the other five tissues. The sequence alignment of putative promoters revealed several variations including SNPs and InDels. These variations resulted in modifications to the corresponding cis-acting elements, forming distinct transcription factor binding sites suggesting the transcriptional-level regulation for these five genes in pearl millet.
CONCLUSIONS
The current study utilized a genome-wide computational analysis to characterize the phospholipase gene family in pearl millet. A comprehensive expression profile of 44 phospholipases led to the identification of five grain-specific candidates. This underscores a potential role for at least these five genes in grain quality traits including the regulation of rancidity in pearl millet. Therefore, this study marks the first exploration highlighting the possible impact of phospholipases towards enhancing agronomic traits in pearl millet.
Topics: Pennisetum; Phospholipases; Multigene Family; Edible Grain; Gene Expression Regulation, Plant; Promoter Regions, Genetic; Phylogeny; Plant Proteins; Synteny; Gene Expression Profiling; Genotype; Chromosome Mapping
PubMed: 38858648
DOI: 10.1186/s12864-024-10504-x -
BioRxiv : the Preprint Server For... Jun 2024The nuclear receptor Liver Receptor Homolog-1 (LRH-1, ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen...
The nuclear receptor Liver Receptor Homolog-1 (LRH-1, ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen unexpectedly identified bilirubin, the product of liver heme metabolism, as a possible ligand for LRH-1. Here, we show unconjugated bilirubin directly binds LRH-1 with apparent =9.3uM, altering LRH-1 interaction with all transcriptional coregulator peptides tested. Bilirubin decreased LRH-1 protease sensitivity, consistent with MD simulations predicting bilirubin stably binds LRH-1 within the canonical ligand binding site. Bilirubin activated a luciferase reporter specific for LRH-1, dependent on co-expression with the bilirubin membrane transporter , but bilirubin failed to activate ligand-binding genetic mutants of LRH-1. Gene profiling in HepG2 cells shows bilirubin selectively regulated transcripts from endogenous LRH-1 ChIP-seq target genes, which was significantly attenuated by either genetic knockdown of LRH-1, or by a specific chemical competitor of LRH-1. Gene set enrichment suggests bilirubin and LRH-1 share roles in cholesterol metabolism and lipid efflux, thus we propose a new role for LRH-1 in directly sensing intracellular levels of bilirubin.
PubMed: 38853895
DOI: 10.1101/2024.05.05.592606 -
Phytomedicine : International Journal... Jul 2024Myocardial infarction (MI) is a serious cardiovascular disease, which presents different pathophysiological changes with the prolongation of the disease. Compound...
BACKGROUND
Myocardial infarction (MI) is a serious cardiovascular disease, which presents different pathophysiological changes with the prolongation of the disease. Compound danshen dripping pills (CDDP) has obvious advantages in MI treatment and widely used in the clinic. However, the current studies were mostly focused on the endpoint of CDDP intervention, lacking the dynamic attention to the disease process. It is of great value to establish a dynamic research strategy focused on the changes in pharmacodynamic substances for guiding clinical medication more precisely.
PURPOSE
It is aimed to explore the dynamic regulating pattern of CDDP on MI based on metabolic trajectory analysis, and then clarify the variation characteristic biomarkers and pharmacodynamic substances in the intervention process.
METHODS
The MI model was successfully prepared by coronary artery left anterior descending branch ligation, and then CDDP intervention was given for 28 days. Endogenous metabolites and the components of CDDP in serum were measured by LC/MS technique simultaneously to identify dynamic the metabolic trajectory and screen the characteristic pharmacodynamic substances at different points. Finally, network pharmacology and molecular docking techniques were used to simulate the core pharmacodynamic substances and core target binding, then validated at the genetic and protein level by Q-PCR and western blotting technology.
RESULTS
CDDP performed typical dynamic regulation features on metabolite distribution, biological processes, and pharmacodynamic substances. During 1-7 days, it mainly regulated lipid metabolism and inflammation, the Phosphatidylcholine (PC(18:1(9Z/18:1(9Z)) and Sphingomyelin (SM(d18:1/23:1(9Z)), SM(d18:1/24:1(15Z)), SM(d18:0/16:1(9Z))) were the main characteristic biomarkers. Lipid metabolism was the mainly regulation pathway during 14-21 days, and the characteristic biomarkers were the Lysophosphatidylethanolamine (LysoPE(0:0/20:0), PE-NMe2(22:1(13Z)/15:0)) and Sphingomyelin (SM(d18:1/23:1(9Z))). At 28 days, in addition to inflammatory response and lipid metabolism, fatty acid metabolism also played the most important role. Correspondingly, Lysophosphatidylcholine (LysoPC(20:0/0:0)), Lysophosphatidylserine (LPS(18:0/0:0)) and Fatty acids (Linoelaidic acid) were the characteristic biomarkers. Based on the results of metabolite distribution and biological process, the characteristic pharmacodynamic substances during the intervention were further identified. The results showed that various kinds of Saponins and Tanshinones as the important active ingredients performed a long-range regulating effect on MI. And the other components, such as Tanshinol and Salvianolic acid B affected Phosphatidylcholine and Sphingomyelin through Relaxin Signaling pathway during the early intervention. Protocatechualdehyde and Rosmarinic acid affected Lysophosphatidylethanolamine and Sphingomyelin through EGFR Tyrosine kinase inhibitor resistance during the late intervention. Tanshinone IIB and Isocryptotanshinone via PPAR signaling pathway affected Lysophosphatidylcholine, Lysophosphatidylserine, and Fatty acids.
CONCLUSION
The dynamic regulating pattern was taken as the entry point and constructs the dynamic network based on metabolic trajectory analysis, establishes the dynamic correlation between the drug-derived components and the endogenous metabolites, and elucidates the characteristic biomarkers affecting the changes of the pharmacodynamic indexes, systematically and deeply elucidate the pharmacodynamic substance and mechanism of CDDP on MI. It also enriched the understanding of CDDP and provided a methodological reference for the dynamic analysis of complex systems of TCM.
Topics: Drugs, Chinese Herbal; Salvia miltiorrhiza; Molecular Docking Simulation; Myocardial Infarction; Animals; Male; Network Pharmacology; Rats, Sprague-Dawley; Biomarkers; Rats; Lysophosphatidylcholines; Camphanes; Panax notoginseng
PubMed: 38850631
DOI: 10.1016/j.phymed.2024.155626 -
Ugeskrift For Laeger May 2024Individuals with antiphospholipid syndrome (APS) have antibodies directed against phospholipid-binding proteins (aPL). The condition is most associated with an increased... (Review)
Review
Individuals with antiphospholipid syndrome (APS) have antibodies directed against phospholipid-binding proteins (aPL). The condition is most associated with an increased risk of thromboembolism and obstetric complications. The 2023 classification criteria for APS include six clinical domains (venous thromboembolism, arterial thrombosis, microvascular events, obstetric events, cardiac valve, thrombocytopaenia) and two laboratory domains (lupus anticoagulant, and anti-cardiolipin or anti-β2-glycoprotein-I antibodies). Diagnosis and treatment of APS are specialist tasks and are summarised in this review.
Topics: Antiphospholipid Syndrome; Humans; Antibodies, Antiphospholipid; Pregnancy; Female; Anticoagulants; Thrombosis
PubMed: 38847311
DOI: 10.61409/V11230715 -
Clinical and Translational Science Jun 2024Homozygous familial hypercholesterolemia (HoFH) is a rare and serious genetic condition characterized by premature cardiovascular disease due to severely elevated... (Review)
Review
Homozygous familial hypercholesterolemia (HoFH) is a rare and serious genetic condition characterized by premature cardiovascular disease due to severely elevated low-density lipoprotein cholesterol (LDL-C). HoFH primarily results from loss-of-function (LOF) mutations in the LDL receptor (LDLR), reducing LDL-C clearance such that patients experience severe hypercholesterolemia, exacerbating the risk of developing cardiovascular events. Treatment options such as statins, lomitapide, ezetimibe, proprotein convertase subtilisin/kexin type 9 inhibitors, and apheresis help lower LDL-C; however, many patients with HoFH still fail to reach their target LDL-C levels and many of these lipid-lowering therapies are not indicated for pediatric use. Angiopoietin-like protein 3 (ANGPTL3) has been identified as a target to treat elevated LDL-C by acting as a natural inhibitor of lipoprotein lipase (LPL) and endothelial lipase (EL), enzymes involved in the hydrolysis of the triglyceride and phospholipid content of very low-density lipoproteins. Persons heterozygous for LOF mutations in ANGPTL3 were reported to have lower LDL-C than non-carriers and lower risk of coronary artery disease. Evinacumab is a first-in-class human monoclonal antibody that specifically binds to ANGPTL3 to prevent its inhibition of LPL and EL. In clinical trials, a 15 mg/kg intravenous dose every 4 weeks has shown a mean percent change from baseline in LDL-C of ~50% in adult, adolescent, and pediatric patients with HoFH. This mini review article describes the mechanism of action of evinacumab, evinacumab population PK and PD modeling, and clinical development history of evinacumab for the treatment of HoFH.
Topics: Humans; Translational Research, Biomedical; Hyperlipoproteinemia Type II; Angiopoietin-Like Protein 3; Cholesterol, LDL; Antibodies, Monoclonal; Broadly Neutralizing Antibodies; Animals; Anticholesteremic Agents; Receptors, LDL
PubMed: 38845393
DOI: 10.1111/cts.13836