Tingting Xu

Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 Å resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.

The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B

In the past 50 years there have been considerable efforts to identify the cellular receptor of hepatitis B virus (HBV). Recently, in vitro evidence from several groups has shown that the sodium–taurocholate cotransporting polypeptide (NTCP, which is encoded by SLC10A1 and transports bile acids into hepatic cells in enterohepatic recirculation) is a strong candidate. In particular, in vitro the p.Ser267Phe variation of SLC10A1 results in loss of HBV receptor function. We tested the role of NTCP as a receptor for HBV in chronic hepatitis B patients using a genetic association study. We selected SLC10A1 variants from 189 exomes. We used Sanger sequencing to follow up the association of the various SLC10A1 variants in a Han Chinese cohort of 1899 chronic hepatitis B patients and 1828 healthy controls. We further investigated the potential impact of the p.Ser267Phe variant on NTCP function using structural analysis. The p.Ser267Phe variant was associated with healthy status (Pu2009=u20095.7 × 10−23, odds ratiou2009=u20090.36) irrespective of hepatitis B virus surface antibody status (Pu2009=u20096.2 × 10−21 and 1.5 × 10−10, respectively, when the cases were compared with hepatitis B virus surface antibody–positive and –negative controls). The variation was also associated with a lower incidence of acute‐on‐chronic liver failure (Pu2009=u20090.007). The estimated heritability explained by this single variation was ∼3.2%. The population prevented fraction was around 13.0% among the southern Chinese. Our structural modeling showed that the p.Ser267Phe variant might interfere with ligand binding, thereby preventing HBV from cellular entry. Conclusion: The p.Ser267Phe NTCP variant is significantly associated with resistance to chronic hepatitis B and a lower incidence of acute‐on‐chronic liver failure. Our results support that NTCP is a cellular receptor for HBV in human infection. (Hepatology 2015;61:1251–1260)

Rare inborn errors associated with chronic hepatitis B virus infection

Chronic hepatitis B (CHB) is a major global health issue. The role of rare genetic variants in CHB has not been elucidated. We aimed to identify rare allelic variants predisposing to CHB. We performed exome sequencing in 50 CHB patients who had no identifiable risk factors for CHB and 40 controls who were healthy and hepatitis B surface antibody‐positive, but had never received hepatitis B vaccination. We selected six rare variant alleles and followed up their association with disease status by Sanger sequencing in a case‐control study comprising 1,728 CHB patients and 1,636 healthy controls. The latter had either not been immunized with hepatitis B vaccine or had uncertain vaccination status. Our results showed that transmembrane protein 2 p.Ser1254Asn, interferon alpha 2 p.Ala120Thr, its regulator NLR family member X1 p.Arg707Cys, and complement component 2 p.Glu318Asp were associated with CHB, with P values of <1.0 × 10−7, 2.76 × 10−5, 5.08 × 10−5, 2.78 × 10−4 and odds ratios (ORs) of 2.45, 4.08, 2.34, and 1.97, respectively. The combined P value was <2.0 × 10−16. As there has been no indication of immunological functions for the associated gene, transmembrane protein 2, we further studied its expression by immunohistochemistry, real‐time polymerase chain reaction, and western blotting. Our results showed that it was strongly expressed by healthy hepatocytes, but its expression was reduced in liver tissues with CHB, hepatitis B viral (HBV) genome‐containing HepG2.2.15 cells, as compared with healthy liver tissues and non‐HBV genome‐containing HepG2 cells (P = 0.022 and 0.0036, respectively). Conclusion: We identified four missense mutations associated with CHB, our results providing evidence for rare inborn genetic defects that contribute to increased host susceptibility to CHB. (HEPATOLOGY 2012;56:1661–1670)

Structural insights into the binding mechanism of IDO1 with hydroxylamidine based inhibitor INCB14943

IDO1 (indoleamine 2, 3-dioxygenase 1), a well characterized immunosuppressive enzyme, has attracted growing attention as a potential target for cancer immunotherapy. Hydroxylamidine compounds INCB024360 and INCB14943 (INCB024360 analogue) are highly effective IDO1 inhibitors. INCB024360 is undergoing clinical trials for treatment of various types of human cancer. Here, we determined the co-crystal structure of IDO1 and INCB14943, and elucidate the detailed binding mode. INCB14943 binds to heme iron in IDO1 protein through the oxime nitrogen. Further analysis also reveals that a halogen bonding interaction between the chlorine atom (3-Cl) of INCB14943 and the sulphur atom of C129 significantly improves the inhibition activity against IDO1. Comparing with the other reported inhibitors, the oxime nitrogen and halogen bond interaction are identified as the unique features of INCB14943 among the IDO1 inhibitors. Thus, our study provides novel insights into the interaction between a small molecule inhibitor INCB14943 and IDO1 protein. The structural information will facilitate future IDO1 inhibitor design.

Structure of Sorting Nexin 11 (SNX11) Reveals a Novel Extended Phox Homology (PX) Domain Critical for Inhibition of SNX10-induced Vacuolation

Background: SNX10 and SNX11 exhibit antagonistic activity in regulating endosome vacuolation. Results: Two additional α-helices downstream of the conventional PX domain were identified in the SNX11 crystal structure. Conclusion: SNX11 contains an extended PX (PXe) domain critical for the inhibition of vacuolation induced by SNX10. Significance: This study reports a novel PXe domain that may represent a new subgroup of the PX domain. Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

Expression, purification, and refolding of active human and mouse secreted group IIE phospholipase A2

Secreted phospholipase A₂s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A₂ (sPLA₂-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA₂-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA₂-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA₂-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA₂-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA₂-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA₂-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA₂-IIEs.

Structure of human SNX10 reveals insights into its role in human autosomal recessive osteopetrosis.

Sorting nexin 10 (SNX10), the unique member of the SNX family having vacuolation activity in cells, was shown to be involved in the development of autosomal recessive osteopetrosis (ARO) in recent genetic studies. However, the molecular mechanism of the disease‐related mutations affecting the biological function of SNX10 is unclear. Here, we report the crystal structure of human SNX10 to 2.6Å resolution. The structure reveals that SNX10 contains the extended phox‐homology domain we previously proposed. Our study provides the structural details of those disease‐related mutations. Combined with the vacuolation study of those mutations, we found that Tyr32 and Arg51 are important for the protein stability and both play a critical role in vacuolation activity, while Arg16Leu may affect the function of SNX10 in osteoclast through protein–protein interactions. Proteins 2014; 82:3483–3489.

Dent Disease in Chinese Children and Findings from Heterozygous Mothers: Phenotypic Heterogeneity, Fetal Growth, and 10 Novel Mutations

OBJECTIVEnTo characterize the phenotypes of Dent disease in Chinese children and their heterozygous mothers and to establish genetic diagnoses.nnnSTUDY DESIGNnUsing a modified protocol, we screened 1288 individuals with proteinuria. A diagnosis of Dent disease was established in 19 boys from 16 families by the presence of loss of function/deleterious mutations in CLCN5 or OCRL1. We also analyzed 16 available patients mothers and examined their pregnancy records.nnnRESULTSnWe detected 14 loss of function/deleterious mutations of CLCN5 in 15 boys and 2 mutations of OCRL1 in 4 boys. Of the patients, 16 of 19 had been wrongly diagnosed with other diseases and 11 of 19 had incorrect or unnecessary treatment. None of the patients, but 6 of 14 mothers, had nephrocalcinosis or nephrolithiasis at diagnosis. Of the patients, 8 of 14 with Dent disease 1 were large for gestational age (>90th percentile); 8 of 15 (53.3%) had rickets. We also present predicted structural changes for 4 mutant proteins.nnnCONCLUSIONSnPediatric Dent disease often is misdiagnosed; genetic testing achieves a correct diagnosis. Nephrocalcinosis or nephrolithiasis may not be sensitive diagnostic criteria. We identified 10 novel mutations in CLCN5 and OCRL1. The possibility that altered CLCN5 function could affect fetal growth and a possible link between a high rate of rickets and low calcium intake are discussed.

A novel mutation, c.494C>A (p.Ala165Asp), in the GPR143 gene causes a mild phenotype in a Chinese X-linked ocular albinism patient.

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Seven Novel and Three Known Mutations in FOXL2 in 10 Chinese Families with Blepharophimosis Syndrome

BACKGROUNDnBlepharophimosis syndrome (BPES) is characterized by eyelid malformation with occasional premature ovarian failure. Mutations in FOXL2 underlie a fraction of BPES cases.nnnOBJECTIVEnWe aimed to investigate the genetic basis of BPES in 26 Chinese families that included 78 patients.nnnMETHODSnWe performed ophthalmological examinations on each family member. We used Sanger sequencing to screen FOXL2 exons and their flanking sequences. We also performed bioinformatics studies, structural modeling and pathogenicity evaluations on all identified variations. Literature was reviewed and genotype-phenotype correlation analysis was performed.nnnRESULTSnThe patients had typical manifestations of BPES. Ten mutations were identified in ten of the twenty-six families. Among these, seven were novel mutations. These included the six truncating mutations, p.Glu69*, p.Gly256Glyfs*14, p.Ala14Serfs*135, p.Pro333Profs*200, p.Pro290Leufs*70, and p.Pro157Profs*91, and one missense mutation, p.Tyr59Cys. The mutations were scattered within the gene, and no mutational hotspots were found. Genotype-phenotype correlation analysis showed that frameshift or nonsense mutations were correlated with type I BPES, while in-frame or missense mutations were associated with type II BPES.nnnCONCLUSIONnWe report the largest BPES cohort in China thus far as well as seven novel mutations in FOXL2. The identification of novel mutations has not only expanded the mutational spectrum of the gene (which is valuable for mutation detection-based screening) but also suggests that most mutations within the Chinese population may not have been characterized yet.