ZFYVE19 deficiency: a ciliopathy involving failure of cell division, with cell death.

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase.

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

Biallelic loss-of-function ZFYVE19 mutations are associated with congenital hepatic fibrosis, sclerosing cholangiopathy and high-GGT cholestasis.

BACKGROUND: For many children with intrahepatic cholestasis and high-serum gamma-glutamyl transferase (GGT) activity, a genetic aetiology of hepatobiliary disease remains undefined. We sought to identify novel genes mutated in children with idiopathic high-GGT intrahepatic cholestasis, with clinical, histopathological and functional correlations. METHODS: We assembled a cohort of 25 children with undiagnosed high-GGT cholestasis and without clinical features of biliary-tract infection or radiological features of choledochal malformation, sclerosing cholangitis or cholelithiasis. Mutations were identified through whole-exome sequencing and targeted Sanger sequencing. We reviewed histopathological findings and assessed phenotypical effects of ZFYVE19 deficiency in cultured cells by immunofluorescence microscopy. RESULTS: Nine Han Chinese children harboured biallelic, predictedly complete loss-of-function pathogenic mutations in ZFYVE19 (c.314C>G, p.S105X; c.379C>T, p.Q127X; c.514C>T, p.R172X; c.547C>T, p.R183X; c.226A>G, p.M76V). All had portal hypertension and, at liver biopsy, histopathological features of the ductal plate malformation (DPM)/congenital hepatic fibrosis (CHF). Four children required liver transplantation for recurrent gastrointestinal haemorrhage. DPM/CHF was confirmed at hepatectomy, with sclerosing small-duct cholangitis. Immunostaining for two primary-cilium axonemal proteins found expression that was deficient intraluminally and ectopic within cholangiocyte cytoplasm. ZFYVE19 depletion in cultured cells yielded abnormalities of centriole and axoneme. CONCLUSION: Biallelic ZFYVE19 mutations can lead to high-GGT cholestasis and DPM/CHF in vivo. In vitro, they can lead to centriolar and axonemal abnormalities. These observations indicate that mutation in ZFYVE19 results, through as yet undefined mechanisms, in a ciliopathy.

Structure of the mouse acidic amino acid decarboxylase GADL1.

Pyridoxal 5'-phosphate (PLP) is a ubiquitous cofactor in various enzyme classes, including PLP-dependent decarboxylases. A recently discovered member of this class is glutamic acid decarboxylase-like protein 1 (GADL1), which lacks the activity to decarboxylate glutamate to γ-aminobutyrate, despite its homology to glutamic acid decarboxylase. Among the acidic amino acid decarboxylases, GADL1 is most similar to cysteine sulfinic acid decarboxylase (CSAD), but the physiological function of GADL1 is unclear, although its expression pattern and activity suggest a role in neurotransmitter and neuroprotectant metabolism. The crystal structure of mouse GADL1 is described, together with a solution model based on small-angle X-ray scattering data. While the overall fold and the conformation of the bound PLP are similar to those in other PLP-dependent decarboxylases, GADL1 adopts a more loose conformation in solution, which might have functional relevance in ligand binding and catalysis. The structural data raise new questions about the compactness, flexibility and conformational dynamics of PLP-dependent decarboxylases, including GADL1.

Splicing analysis of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase.

AIM: The aim of this study was to analyze the pathogenicity of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase. METHODS: The enrolled variants were identified in ABCB11 between October 2009 and June 2016. The effects on pre-RNA splicing were analyzed by in silico tools and minigene splicing assay. RESULTS: There were three intronic (c.908 + 5G > A, c.2815-8A > G, and c.612-15_-6del10bp) and two synonymous (c.1809G > A, p.K603 K and c.2418C > T, p.G806G) variants with unknown significance identified in ABCB11 of five ABCB11 heterozygotes. Parental studies were carried out for four patients, and revealed that the variants with unknown significance were compound heterozygous with other pathogenic variants. The five variants with unknown significance had minor allele frequency <0.1% or were absent from controls, and had positive prediction results by in silico tools. The effects on pre-RNA splicing were further confirmed by minigene splicing assay. c.908 + 5A caused abnormal splicing in at least 78.5 ± 3.8% of products using a cryptic splice site (ss) 22 nucleotides (nt) upstream of the wild-type (WT) 5'ss. Seven nucleotides of intron 22 upstream of the WT 3'ss was retained for all products from c.2815-8G. c.612-15_-6del caused exon 8 skipping in 24.8 ± 7.7% of products, and 55 nt of exon 8 downstream of the WT 3'ss removal in remaining products. c.1809A led to exon 15 skipping. c.2418 T removed exon 20 and 62 nt of exon 21 downstream of the WT 3'ss by using a cryptic ss. CONCLUSIONS: We successfully identified five pathogenic synonymous or intronic variants with some common features. These features might help to choose the right variant for further functional assay.

Novel NBAS mutations and fever-related recurrent acute liver failure in Chinese children: a retrospective study.

BACKGROUND: Underlying causes in Chinese children with recurrent acute liver failure (RALF), including liver crises less than full acute liver failure, are incompletely understood. We sought to address this by searching for genes mutated in such children. METHODS: Five unrelated Chinese boys presenting between 2012 and 2015 with RALF of unexplained etiology were studied. Results of whole exome sequencing were screened for mutations in candidate genes. Mutations were verified in patients and their family members by Sanger sequencing. All 5 boys underwent liver biopsy. RESULTS: NBAS was the only candidate gene mutated in more than one patient (biallelic mutations, 3 of 5 patients; 5 separate mutations). All NBAS mutations were novel and predictedly pathogenic (frameshift insertion mutation c.6611_6612insCA, missense mutations c.2407G > A and c.3596G > A, nonsense mutation c.586C > T, and splicing-site mutation c.5389 + 1G > T). Of these mutations, 3 lay in distal (C-terminal) regions of NBAS, a novel distribution. Unlike the 2 patients without NBAS mutations, the 3 patients with confirmed NBAS mutations all suffered from a febrile illness before each episode of liver crisis (fever-related RALF), with markedly elevated alanine aminotransferase and aspartate aminotransferase activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy and mild to moderate jaundice. CONCLUSIONS: As in other countries, so too in China; NBAS disease is a major cause of fever-related RALF in children. The mutation spectrum of NBAS in Chinese children seems different from that described in other populations.

Recombinant portal protein from Staphylococcus epidermidis bacteriophage CNPH82 is a 13-subunit oligomer.

The portal protein cn3 of bacteriophage CNPH82 is predicted to serve as a gateway for translocation of viral genome into preformed pro-capsid, like portal proteins from other double-stranded DNA tailed bacteriophages. The host of bacteriophage CNPH82 is the opportunistic human pathogenic bacterium Staphylococcus epidermidis, a major cause of nosocomial infections. The portal protein of this phage has been cloned, overexpressed and purified. Size-exclusion chromatography-multi-angle laser light scattering analysis has indicated that the portal protein contains ∼13 subunits. Crystals of the portal protein, diffracting to 4.2 Å, have been obtained. These crystals belong to the space group C222(1) with the unit-cell parameters of a = 252.4, b = 367.0, c = 175.5 Å. The self-rotation function revealed the presence of a single 13-subunit oligomer in the asymmetric unit.

Trp RNA-binding attenuation protein: modifying symmetry and stability of a circular oligomer.

BACKGROUND: Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins. METHODOLOGY/PRINCIPAL FINDINGS: Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts--have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP. CONCLUSIONS/SIGNIFICANCE: We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

Three isoforms of anti-lipopolysaccharide factor identified from eyestalk cDNA library of swimming crab Portunus trituberculatus.

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In this study, three isoforms of the ALF homologues (PtesALF1-3) were identified from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA sequences of PtesALF1, 2 and 3 were 1138, 1052 and 1057 bp encoding 92, 108 and 123 amino acids, respectively. PtesALF1-3 contained two conserved cysteine residues and shared high similarity with other reported ALFs. Predicted tertiary structures of PtesALF2 and 3 containing four ß-strands and three α-helix were similar to that described in Limulus polyphemus, while PtesALF1 had only one α-helix in its spatial structure. Sequence analysis revealed PtesALF1-3 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Totally 89 SNPs including 18 in coding region and 71 in noncoding region were detected by direct sequencing of 30 genomic samples. The mRNA expression of PtesALF1 and PtesALF1-3 transcripts was mainly detected in haemocytes but showed different expression pattern in other tissues including hepatopancreas, gill, eyestalk and muscle. After challenge with Vibrio alginolyticus, the temporal expression level of PtesALF1-3 transcripts in haemocytes showed a clear time-dependent response expression pattern with two peaks within the experimental period of 32 h, while PtesALF1 was up-regulated only once with obvious decrease at 6 h and significant increase at 24 h. These results suggest that the PtesALF isoforms have different tissue specificity and might provide multiple protective functions against invading bacteria in P. trituberculatus.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus.

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab.

Molecular cloning and characterization of a heat shock protein 70 gene in swimming crab (Portunus trituberculatus).

Hsp70 can stimulate cells of the innate immune system directly by acting as "danger"-signaling molecules. To understand the immune defense mechanisms of swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae), the cDNA of Hsp70 (designated PtHsp70) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The full-length PtHsp70 cDNA was 2195 bp, including an open reading frame (ORF) of 1950 bp encoding a polypeptide of 650 amino acids with estimated molecular mass of 71.1514 kDa and theoretical isoelectric point of 5.38. FASTA and BLAST analysis indicated that PtHsp70 should be an inducible cytosolic member of the Hsp70 family. The coding region of PtHsp70 was uninterrupted and four SNPs with 1133C/T, 1311C/T, 1551C/T and 1809 A/G were detected by direct sequencing of 20 genomic samples. Using fluorescent real-time quantitative PCR, the transcriptional expression of PtHsp70 showed a clear time-dependent response after challenge by Vibrio alginolyticus, the main causative agent of emulsification disease causing large mortality in P. trituberculatus. This is the first report on the expression of Hsp70 induced by pathogen stimulation in Brachyura. Phylogenetic analysis revealed that the inducible Hsp70s were divided into two groups in crab and PtHsp70 was clustered into the Hsp/Hsc group (Clade I) by maximum-likelihood (ML) and Bayesian inference (BI) methods. GAP repeat and GGMP motif of inducible Hsp70 gene in the crab species were only found in Clade I.