Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas,Vanua-Lava and Viangchan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.     

The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k_cat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T₅₀ (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP⁺ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP⁺, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP⁺, leading to enzyme instability.     

Small-Molecule Activators of Glucose-6-phosphate Dehydrogenase (G6PD) Bridging the Dimer Interface

We recently identified AG1, a small-molecule activator that functions by promoting oligomerization of glucose-6-phosphate dehydrogenase (G6PD) to the catalytically competent forms. Biochemical experiments indicate that the activation of G6PD by the original hit molecule (AG1) is noncovalent and that one C₂-symmetric region of the G6PD homodimer is important for ligand function. Consequently, the disulfide in AG1 is not required for activation of G6PD, and a number of analogues were prepared without this reactive moiety. Our study supports a mechanism of action whereby AG1 bridges the dimer interface at the structural nicotinamide adenine dinucleotide phosphate (NADP⁺) binding sites of two interacting G6PD monomers. Small molecules that promote G6PD oligomerization have the potential to provide a first-in-class treatment for G6PD deficiency. This general strategy could be applied to other enzyme deficiencies in which control of oligomerization can enhance enzymatic activity and/or stability.     

Cardiolipin Stabilizes and Increases Catalytic Efficiency of Carnitine Palmitoyltransferase II and Its Variants S113L,P50H,and Y479F

Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in K_M. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.     

新型罗丹明B衍生物的合成及其对Cu^2＋的检测

以罗丹明6G、乙二胺、水杨醛为原料在无水乙醇中合成了一种新型罗丹明B类衍生物。在pH=6.54条件下,该衍生物的乙醇溶液无荧光及紫外-可见吸收。相比于其它一些常见金属离子,其对Cu（Ⅱ）具有较高的选择性响应：加入Cu2＋溶液后,溶液迅速由无色变为粉红色,表现出较大的荧光强度和紫外-可见吸收且结合过程为可逆反应。由该衍生物对Cu（Ⅱ）响应的工作曲线得出：Cu（Ⅱ）浓度在0~1.0×10-4mol/L范围内服从朗伯-比尔定律,相关系数r=0.9973,检出限为6.328×10-8μg/mL。应用等摩尔连续变化法求得该衍生物与Cu（Ⅱ）络合比为1：1,条件稳定常数K=2.561×105（室温）。     

Does Prop-2-ynylideneamine,HC≡CCH=NH,Exist in Space? A Theoretical and Computational Investigation

MP2, DFT and CCSD methods with 6-311++G** and aug-cc-pvdz basis sets have been used to probe the structural changes and relative energies of E-prop-2-ynylideneamine (I), Z-prop-2-ynylideneamine (II), prop-1,2-diene-1-imine (III) and vinyl cyanide (IV). The energy near-equivalence and provenance of preference of isomers and tautomers were investigated by NBO calculations using HF and B3LYP methods with 6-311++G** and aug-cc-pvdz basis sets. All substrates have Cs symmetry. The optimized geometries were found to be mainly theoretical method dependent. All elected levels of theory have computed I/II total energy of isomerization (ΔE) of 1.707 to 3.707 kJ/mol in favour of II at 298.15 K. MP2 and CCSD methods have indicated clearly the preference of II over III; while the B3LYP functional predicted nearly similar total energies. All tested levels of theory yielded a global II/IV tautomerization total energy (ΔE) of 137.3–148.4 kJ/mol in support of IV at 298.15 K. The negative values of ΔS indicated that IV is favoured at low temperature. At high temperature, a reverse tautomerization becomes spontaneous and II is preferred. The existence of II in space was debated through the interpretation and analysis of the thermodynamic and kinetic studies of this tautomerization reaction and the presence of similar compounds in the Interstellar Medium (ISM).     

Characterization of Novel Pathogenic Variants Causing Pyridox(am)ine 5′-Phosphate Oxidase-Dependent Epilepsy

Several variants of the enzyme pyridox(am)ine 5′-phosphate oxidase (PNPO), responsible for a rare form of vitamin B₆-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5′-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5′-phosphate product.     

tert-Butyl Hydroperoxide (tBHP)-Induced Lipid Peroxidation and Embryonic Defects Resemble Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in C. elegans

G6PD is required for embryonic development in animals, as severe G6PD deficiency is lethal to mice, zebrafish and nematode. Lipid peroxidation is linked to membrane-associated embryonic defects in Caenorhabditis elegans (C. elegans). However, the direct link between lipid peroxidation and embryonic lethality has not been established. The aim of this study was to delineate the role of lipid peroxidation in gspd-1-knockdown (ortholog of g6pd) C. elegans during reproduction. tert-butyl hydroperoxide (tBHP) was used as an exogenous inducer. Short-term tBHP administration reduced brood size and enhanced germ cell death in C. elegans. The altered phenotypes caused by tBHP resembled GSPD-1 deficiency in C. elegans. Mechanistically, tBHP-induced malondialdehyde (MDA) production and stimulated calcium-independent phospholipase A₂ (iPLA) activity, leading to disturbed oogenesis and embryogenesis. The current study provides strong evidence to support the notion that enhanced lipid peroxidation in G6PD deficiency promotes death of germ cells and impairs embryogenesis in C. elegans.     

Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.     

GBA Mutations Influence the Release and Pathological Effects of Small Extracellular Vesicles from Fibroblasts of Patients with Parkinson’s Disease

Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson’s disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal–lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.     

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions

The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.     

In Silico Analysis of the Molecular-Level Impact of SMPD1 Variants on Niemann-Pick Disease Severity

Sphingomyelin phosphodiesterase (SMPD1) is a key enzyme in the sphingolipid metabolism. Genetic SMPD1 variants have been related to the Niemann-Pick lysosomal storage disorder, which has different degrees of phenotypic severity ranging from severe symptomatology involving the central nervous system (type A) to milder ones (type B). They have also been linked to neurodegenerative disorders such as Parkinson and Alzheimer. In this paper, we leveraged structural, evolutionary and stability information on SMPD1 to predict and analyze the impact of variants at the molecular level. We developed the SMPD1-ZooM algorithm, which is able to predict with good accuracy whether variants cause Niemann-Pick disease and its phenotypic severity; the predictor is freely available for download. We performed a large-scale analysis of all possible SMPD1 variants, which led us to identify protein regions that are either robust or fragile with respect to amino acid variations, and show the importance of aromatic-involving interactions in SMPD1 function and stability. Our study also revealed a good correlation between SMPD1-ZooM scores and in vitro loss of SMPD1 activity. The understanding of the molecular effects of SMPD1 variants is of crucial importance to improve genetic screening of SMPD1-related disorders and to develop personalized treatments that restore SMPD1 functionality.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

青霉烷酸二苯甲酯亚砜的合成方法探索

由6-APA合成了他唑巴坦的重要中间体--青霉烷酸二苯甲酯亚砜.在氧化反应中,使用双氧水作氧化剂,在消去溴的反应中,使用铝粉代替锌粉,合成了目标化合物,三步总收率为60.7%,为该化合物的合成提供了新的路线.     

Computational Insights into the Deleterious Impacts of Missense Variants on N-Acetyl-d-glucosamine Kinase Structure and Function

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.     

Einfluß von Lebertrangaben auf die Funktion der Lebermitochondrien in Langzeitfütterungsstudien an Ratten

Influence of n-3 Fatty Acids on Mitochondrial Function and Stability of Erythrocyte Membrane of Rats in Long Term Experiments with Cod Liver Oil The influence of different amounts of polyunsaturated fatty acids (PUFA) (1.3, 2.6 and 6.3% of total energy intake) on mitochondrial respiration and the stability of erythrocyte membrane was tested in experiments with rats lasting 12 and 32 weeks. The fat component of the semisynthetic diets (6g/100 g diet) was made up of coconut fat and cod liver oil (Gr. I, II, III) and cod liver oil and linoleic methylester (G1. IV). The n-3 fatty acids amounted to 1.18 cal% (I), 2.35 cal% (II,III) and 2.1cal% (IV). The diets of the groups I, III and IV wre supplemented with 6 mg D-α-tocopherylequivalents per 100g; the tocopherol/PUFA-ratios (mg/g) in the diets I, II, III, IV were 10.7, 0.1, 5.4 and 2.3 respectively. After 12 weeks cod liver oil had no significant influence on total lipids of the liver, hemolysis rate of red blood cells as well as the respiration of liver mitochondria. Highest weight gained was reported for the animals of group I receiving 1.3 cal% of PUFA derived from cod liver oil. All groups had similar relative liver weights. After 32 weeks the consequences of the insufficient supply tocopherol in group II were a significantly increased hemolysis rate of the erythrocytes and a decreased respiratory control index as well as the ADP/O-ratios of liver mitochondria using succinate and malate/glutamate as substrates. The highest PUFA amount fed (6.3 cal%; derived from cod liver oil and linoleic methylester) with the adequate vit. E supplementation did not cause any major alterations. The results show that fatty acids of the α-linolenic acid group can replace in part the n-6 fatty acid in their essential role for integrity of the membranes of mitochondria and erythrocytes. This is possible only if the increased antioxidant requirement of the body caused by ingestion of fish oil PUFA's is adequately compensated through additional supplements with antioxidants like α-tocopherol.     

Derivatization of keto fatty acids: Part XIII—Synthesis of methyl hexahydro-3-alkyl-6-thioxo-1,2,4,5-tetrazine-3-alkanoates

Hexahydrothioxotetrazine fatty derivatives have been prepared by the reaction of thiocarbohydrazide with oxo fatty esters. Methyl 10-oxoundecanoate (I), methyl 9-oxooctadecanoate (II) and methyl 12-oxooctadecanoate (III) on treatment with thiocarbohydrazide furnish methyl hexahydro-3-methyl-6-thioxo-1,2,4,5-tetrazine-3-nonanoate (IV), methyl hexahydro-3-nonyl-6-thioxo-1,2,4,5-tetrazine-3-octanoate (V) and methyl hexahydro-3-hexyl-6-thioxo-1,2,4,5-tetrazine-3-undecanoate (VI), respectively, in fairly good yields. The structural assignments of the hexahydrothioxotetrazine fatty derivatives are based on microanalysis, infrared, nuclear magnetic resonance and mass spectrometry data.     

Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.)

The three-amino-acid-loop-extension (TALE) superfamily genes broadly existed in plants, which played important roles in plant growth, development and abiotic stress responses. In this study, we identified 68 Glycine max TALE (GmTALE) superfamily members. Phylogenetic analysis divided the GmTALE superfamily into the BEL1-like (BLH/BELL homeodomain) and the KNOX (KNOTTED-like homeodomain) subfamilies. Moreover, the KNOX subfamily could be further categorized into three clades (KNOX Class I, KNOX Class II and KNOX Class III). The GmTALE genes showed similarities in the gene structures in the same subfamily or clade, whose coding proteins exhibited analogous motif and conserved domain compositions. Besides, synteny analyses and evolutionary constraint evaluations of the TALE members among soybean and different species provided more clues for GmTALE superfamily evolution. The cis-element analyses in gene promoter regions and relevant gene expression profiling revealed different regulating roles of GmTALE genes during soybean plant development, saline and dehydration stresses. Genome-wide characterization, evolution, and expression profile analyses of GmTALE genes can pave the way for future gene functional research and facilitate their roles for applications in genetic improvement on soybean in saline and dehydration stresses.