Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

The three-dimensional structures of mutants of porphobilinogen deaminase: toward an understanding of the structural basis of acute intermittent porphyria.

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.     

Recurrent porphyria attacks in a Chinese patient with a heterozygous PBGD mutation

We report here the case of a 32-year-old Chinese Han woman who presented with frequent severe abdominal pain, convulsion, numbness and confusion. She also had hypertension, hyponatremia, chronic renal failure, anemia and a high urinary δ-aminolevulinic acid concentration. We identified a heterozygous splicing mutation in intron 11 (IVS11-2A → G) of the porphobilinogen (PBG) deaminase gene (PBGD) in her genomic DNA. This mutation had previously been reported in a North American patient, but was absent from 50 healthy Chinese controls.     

Subcellular location of the tetrapyrrole synthesis enzyme porphobilinogen deaminase in higher plants: an immunological investigation

A recombinant plasmid, pArab8, harbouring the cDNA encoding the mature form of the tetrapyrrole synthesis enzyme porphobilinogen deaminase (EC 4.3.1.8; also known as hydroxymethylbilane synthase) from Arabidopsis thaliana (L.) Heynh. has been constructed, and used to transform Escherichia coli. The porphobilinogen deaminase protein from Arabidopsis was overexpressed in this strain, and purified to homogeneity (3000-fold) with a yield of 20%. Antibodies were raised against the purified plant enzyme, and used in Western blot analysis, immunoprecipitation of enzyme activity and immuno-gold electron microscopy. The results indicate that the enzyme is confined to plastids in both leaves and roots. The implications of this finding for plant tetrapyrrole synthesis are discussed.Abbreviations DEAE diethylaminoethyl - FPLC fast protein liquid chromatography - PBG porphobilinogen This work was supported by Science and Engineering Research Council (SERC) and Agricultural and Food Research Council (AFRC) grants to P.M.J. and an AFRC grant to A.G.S. The protein sequencing was carried out by Mr Lawrence Hunt of the SERC MRI Protein Sequencing Unit (Director Dr M.G. Gore) at Southampton University. We acknowledge the Wellcome Foundation for financial support of the Protein and Nucleic Acid Chemistry Facility at the University of Cambridge, where the oligonucleotide primers were synthesised.     

Structural basis for dysregulation of aminolevulinic acid synthase in human disease

Heme is a critical biomolecule that is synthesized in vivo by several organisms such as plants, animals, and bacteria. Reflecting the importance of this molecule, defects in heme biosynthesis underlie several blood disorders in humans. Aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in α-proteobacteria and nonplant eukaryotes. Debilitating and painful diseases such as X-linked sideroblastic anemia and X-linked protoporphyria can result from one of more than 91 genetic mutations in the human erythroid-specific enzyme ALAS2. This review will focus on recent structure-based insights into human ALAS2 function in health and how it dysfunctions in disease. We will also discuss how certain genetic mutations potentially result in disease-causing structural perturbations. Furthermore, we use thermodynamic and structural information to hypothesize how the mutations affect the human ALAS2 structure and categorize some of the unique human ALAS2 mutations that do not respond to typical treatments, that have paradoxical in vitro activity, or that are highly intolerable to changes. Finally, we will examine where future structure-based insights into the family of ALA synthases are needed to develop additional enzyme therapeutics.     

l-2-Hydroxyglutarate dehydrogenase: identification of a novel enzyme activity in rat and human liver. Implications for l-2-hydroxyglutaric acidemia

In this paper we studied the degradation of l-2-hydroxyglutarate in tissues from rat and man in order to try and find the underlying basis for the accumulation of this metabolite in l-2-hydroxyglutaric patients. The results show that l-2-hydroxyglutarate is not degraded by an oxidase but via a dehydrogenase which was found to be present in liver only. This newly identified enzyme activity was characterized kinetically, although the nature of the reaction product remains to be identified.     

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.     

Effects of volatile anaesthetics on heme metabolism in a murine genetic model of Acute Intermittent Porphyria. A comparative study with other porphyrinogenic drugs

Background

Acute Intermittent Porphyria (AIP) is an inherited disease produced by a deficiency of Porphobilinogen deaminase (PBG-D). The aim of this work was to evaluate the effects of Isoflurane and Sevoflurane on heme metabolism in a mouse genetic model of AIP to further support our previous proposal for avoiding their use in porphyric patients. A comparative study was performed administering the porphyrinogenic drugs allylisopropylacetamide (AIA), barbital and ethanol, and also between sex and mutation using AIP (PBG-D activity 70% reduced) and T1 (PBG-D activity 50% diminished) mice.

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-β-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.     

Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn²⁺-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD⁺ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the “sandwich structure” on the adenine moiety of NAD⁺. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H₂O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.     

Analysis of severe human adenovirus infection outbreak in Guangdong Province,southern China in 2019

During 2018-2019, a severe human adenovirus (HAdV) infection outbreak occurred in southern China. Here, we screened 18 respiratory pathogens in 1704 children (≤ 14 years old) hospitalized with acute respiratory illness in Guangzhou, China, in 2019. In total, 151 patients had positive HAdV test results; 34.4% (52/151) of them exhibited severe illness. HAdV infection occurred throughout the year, with a peak in summer. The median patient age was 3.0 (interquartile range:1.1-5.0) years. Patients with severe HAdV infection exhibited increases in 12 clinical indexes (P ≤ 0.019) and decreases in four indexes (P ≤ 0.007), compared with patients exhibiting nonsevere infection. No significant differences were found in age or sex distribution according to HAdV infection severity (P > 0.05); however, the distributions of comorbid disease and HAdV co-infection differed according to HAdV infection severity (P < 0.05). The main epidemic types were HAdV-3 (47.0%, 71/151) and HAdV-7 (46.4%, 70/151). However, the severe illness rate was significantly higher in patients with HAdV-7 (51.4%) than in patients with HAdV-3 (19.7%) and other types of HAdV (20%) (P < 0.001). Sequencing analysis of genomes/capsid genes of 13 HAdV-7 isolates revealed high similarity to previous Chinese isolates. A representative HAdV-7 isolate exhibited a similar proliferation curve to the curve described for the epidemic HAdV-3 strain Guangzhou01 (accession no. DQ099432) (P > 0.05); the HAdV-7 isolate exhibited stronger virulence and infectivity, compared with HAdV-3 (P < 0.001). Overall, comorbid disease, HAdV co-infection, and high virulence and infectivity of HAdV-7 were critical risk factors for severe HAdV infection; these data can facilitate treatment, control, and prevention of HAdV infection.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

植物萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原异构酶的分子结构特征与功能预测分析

采用生物信息学的方法和工具对已在GenBank上注册的拟南芥、玉米、岩蔷薇、水稻、黄花蒿、亚麻等植物的萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原异构酶的核酸及氨基酸序列进行分析,并对其组成成分、转运肽、跨膜拓朴结构域、疏水性/亲水性、蛋白质二级及三级结构、分子系统进化关系等进行预测和推断。结果表明：该类酶基因的全长包括5′、3′非翻译区和一个开放阅读框,无跨膜结构域,是一个具转运肽的亲水性蛋白,包括两个功能DXR结合motif及两个功能NADPH结合motif,α-螺旋和不规则卷曲是蛋白质二级结构最大量的结构元件,β-转角和β-折叠散布于整个蛋白质中,蛋白质的功能域在空间结构上折叠成“V”形,“V”形的两臂由N-端与C-端构成,“V”形的底部,是N 端臂与C-端臂的结合域。     

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.     

Structural basis of tumor suppressor in lung cancer 1 (TSLC1) binding to differentially expressed in adenocarcinoma of the lung (DAL-1/4.1B)

Perturbed cell adhesion mechanisms are crucial for tumor invasion and metastasis. A cell adhesion protein, TSLC1 (tumor suppressor in lung cancer 1), is inactivated in a majority of metastatic cancers. DAL-1 (differentially expressed in adenocarcinoma of the lung protein), another tumor suppressor, binds through its FERM domain to the TSLC1 C-terminal, 4.1 glycophorin C-like, cytoplasmic domain. However, the molecular basis for this interaction is unknown. Here, we describe the crystal structure of a complex between the DAL-1 FERM domain and a portion of the TSLC1 cytoplasmic domain. DAL-1 binds to TSLC1 through conserved residues in a well defined hydrophobic pocket in the structural C-lobe of the DAL-1 FERM domain. From the crystal structure, it is apparent that Tyr(406) and Thr(408) in the TSLC1 cytoplasmic domain form the most important interactions with DAL-1, and this was also confirmed by surface plasmon resonance studies. Our results refute earlier exon deletion experiments that indicated that glycophorin C interacts with the α-lobe of 4.1 FERM domains.     

Clinical characteristics and molecular epidemiology of human metapneumovirus in children with acute lower respiratory tract infections in China, 2017 to 2019: A multicentre prospective observational study

Human metapneumovirus (HMPV) infection is one of the leading causes of hospitalization in young children with acute respiratory illness. In this study, we prospectively collected respiratory tract samples from children who were hospitalized with acute lower respiratory tract infection in six hospitals in China from 2017 to 2019. HMPV was detected in 145 out of 2733 samples (5.3%) from the hospitalized children. The majority of HMPV-positive children were under the age of two (67.6%), with a median age of one year. HMPV can independently cause acute lower respiratory tract infection in young children, while all patients showed mild clinical symptoms. Of all the co-infected patients, HMPV was most commonly detected with enterovirus (EV) or rhinovirus (RhV) (38.0%, followed by respiratory syncytial virus (RSV) (32.0%). The highest detection rate occurred from March to May in both northern and southern China. Out of 145 HMPV positive samples, 48 were successfully typed, of which 36 strains were subgrouped into subtypes A2c (75%), eight strains were included in subtype B1 (16.7%), and four strains were included in subtype B2 (8.3%). Moreover, 16 A2c strains contained 111-nucleotide duplications in the G gene. Twenty-seven complete HMPV genomes were successfully obtained, and 25 (92.6%) strains belonged to subtype A2c, whereas one strain was included in subgroup B1 and another was included in subgroup B2. A total of 277 mutations were observed in the complete genomes of 25 A2c strains. All results presented here improve our understanding of clinical characteristics and molecular epidemiology of HMPV infection in children.     

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A₂ kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A₂ kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Galα1–4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of Gaα1–3(Fucα1–2)Galβ-HexNac-Galα1–4Galβ-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant glood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which migh explain the low expression of type-4 chain blood group heptaglycosylceramides in human blood group B kidneys.