Missense mutation Q384K in the APOB gene affecting the large lipid transfer module of apoB reduces the secretion of apoB-100 in the liver without reducing the secretion of apoB-48 in the intestine.

BACKGROUND: Molecular genetic testing of patients with hypobetalipoproteinemia may identify a genetic cause that can form the basis for starting proper therapy. Identifying a genetic cause may also provide novel data on the structure-function relationship of the mutant protein. OBJECTIVE: To identify a genetic cause of hypobetalipoproteinemia in a patient with levels of low density lipoprotein cholesterol at the detection limit of 0.1 mmol/l. METHODS: DNA sequencing of the translated exons with flanking intron sequences of the genes adenosine triphosphate-binding cassette transporter 1, angiopoietin-like protein 3, apolipoprotein B, apolipoprotein A1, lecithin-cholesterol acyltransferase, microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9. RESULTS: The patient was homozygous for mutation Q384K (c.1150C>A) in the apolipoprotein B gene, and this mutation segregated with hypobetalipoproteinemia in the family. Residue Gln384 is located in the large lipid transfer module of apoB that has been suggested to be important for lipidation of apolipoprotein B through interaction with microsomal triglyceride transfer protein. Based on measurements of serum levels of triglycerides and apolipoprotein B-48 after an oral fat load, we conclude that the patient was able to synthesize apolipoprotein B-48 in the intestine in a seemingly normal fashion. CONCLUSION: Our data indicate that mutation Q384K severely reduces the secretion of apolipoprotein B-100 in the liver without reducing the secretion of apolipoprotein B-48 in the intestine. Possible mechanisms for the different effects of this and other missense mutations affecting the large lipid transfer module on the two forms of apoB are discussed.

Lysosomal acid lipase does not have a propeptide and should not be considered being a proprotein.

Lysosomal acid lipase (LAL) plays an important role in lipid metabolism by performing hydrolysis of triglycerides and cholesteryl esters in the lysosome. Based upon characteristics of LAL purified from human liver, it has been proposed that LAL is a proprotein with a 55 residue propeptide that may be essential for proper folding, intracellular transport, or enzymatic function. However, the biological significance of such a propeptide has not been fully elucidated. In this study, we have performed a series of studies in cultured HepG2 and HeLa cells to determine the role of the putative propeptide. However, by Western blot analysis and subcellular fractionation, we have not been able to identify a cleaved LAL lacking the N-terminal 55 residues. Moreover, mutating residues surrounding the putative cleavage site at Lys76 ↓ in order to disrupt a proteinase recognition sequence, did not affect LAL activity. Furthermore, forcing cleavage at Lys76 ↓ by introducing the optimal furin cleavage site RRRR↓EL between residues 76 and 77, did not affect LAL activity. These data, in addition to bioinformatics analyses, indicate that LAL is not a proprotein. Thus, it is possible that the previously reported cleavage at Lys76 ↓ could have resulted from exposure to proteolytic enzymes during the multistep purification procedure.

Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform.

Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3'-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform.

Mutations affecting the transmembrane domain of the LDL receptor: impact of charged residues on the membrane insertion.

Familial hypercholesterolemia (FH) is caused by mutations in the low density lipoprotein receptor (LDLR) gene. To study the impact of mutations affecting the hydrophobic transmembrane domain of the LDLR, each of the 22 amino acids of the transmembrane domain was individually mutated to arginine. The more centrally in the transmembrane domain an arginine was located, the lower amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum were observed. This led to lower amounts of the 160 kDa mature LDLR on the cell surface. For the mutants V797R-LDLR, L798R-LDLR and L799R-LDLR a proportion of full-length receptors including the transmembrane and cytoplasmic domains, was secreted into the endoplasmic reticulum lumen to appear in the culture medium. When the transmembrane domain of the epidermal growth factor receptor (EGFR) was replaced by that of the mutant L799R-LDLR, similar effects were observed for the EGFR as for L799R-LDLR. Introducing arginines in the transmembrane domain of the LDLR also affected metalloproteinase cleavage of the ectodomain and γ-secretase cleavage within the transmembrane domain. The most likely explanation for the low amounts of the 120 kDa precursor is that a basic residue in the hydrophobic transmembrane domain prevents the mutant LDLR from being inserted in the endoplasmic reticulum membrane from the Sec61 translocon complex. As a consequence, quality control systems could be activated. However, our data indicate that proteasomal degradation, lysosomal degradation, autophagy or ectodomain cleavage were not the underlying mechanism for degradation of these mutant LDLRs.

Mutation p.L799R in the LDLR, which affects the transmembrane domain of the LDLR, prevents membrane insertion and causes secretion of the mutant LDLR.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH). The mechanism by which mutations in the LDLR affecting the transmembrane domain of the receptor cause FH has not been thoroughly investigated. In this study, we have selected 12 naturally occurring mutations affecting the transmembrane domain and studied their effect on the LDLR. The main strategy has been to transiently transfect HepG2 cells with mutant LDLR plasmids and to study the mutant LDLRs in cell lysates and in media by western blot analysis. The most striking finding was that mutation p.L799R led to secretion of the entire 160 kDa mature L799R-LDLR. Residue 799Leu is in the middle of the 22-residue transmembrane domain, and introduction of a basic residue in the hydrophobic core of the transmembrane domain could prevent L799R-LDLR from being correctly recognized and integrated in the membrane by the Sec61 translocon complex. This would then lead to translocation of the entire L799R-LDLR into the lumen of the endoplasmic reticulum. Mutation p.L799R should be considered a member of a separate class of FH-causing mutations that affects the insertion of the LDLR in the cell membrane.

Early-onset Crohn's disease and autoimmunity associated with a variant in CTLA-4.

OBJECTIVE: IBD is a group of complex, systemic disorders associated with intestinal inflammation and extraintestinal manifestations. Recent studies revealed Mendelian forms of IBD, which contributed significantly to our understanding of disease pathogenesis and the heritability of IBD. DESIGN: We performed exome sequencing in a family with Crohn's disease (CD) and severe autoimmunity, analysed immune cell phenotype and function in affected and non-affected individuals, and performed in silico and in vitro analyses of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) structure and function. RESULTS: A novel missense variant was identified in CTLA4 encoding CTLA-4, a coinhibitory protein expressed by T cells and required for regulation of T cell activation. The residue affected by the mutation, CTLA-4 Tyr60, is evolutionarily highly conserved, and the identified Y60C variant is predicted to affect protein folding and structural stability and demonstrated to cause impaired CTLA-4 dimerisation and CD80 binding. Intestinal inflammation and autoimmunity in carriers of CTLA-4 Y60C exhibit incomplete penetrance with a spectrum of clinical presentations ranging from asymptomatic carrier status to fatal autoimmunity and intestinal inflammation. In a clinically affected CTLA-4 Y60C carrier, T cell proliferation was increased in vitro and associated with an increased ratio of memory to naive T cells in vivo, consistent with impaired regulation of T cell activation. CONCLUSIONS: Our results support the concept that variants in CTLA4 provide the basis for a novel Mendelian form of early-onset CD associated with systemic autoimmunity. Incomplete penetrance of autoimmunity further indicates the presence of other genetic and/or environmental modifiers.

Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cß, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cß arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cß kinase family is exceptionally high, a small set of signature residues defining Cα and Cß subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cß clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+)/Mn(2+). Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

BACKGROUND & AIMS: A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). METHODS: We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. RESULTS: Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. CONCLUSIONS: We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.

Schizosaccharomyces pombe Ofd2 is a nuclear 2-oxoglutarate and iron dependent dioxygenase interacting with histones.

2-Oxoglutarate (2OG) dependent dioxygenases are ubiquitous iron containing enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. They participate in a wide range of biological processes including collagen biosynthesis, fatty acid metabolism, hypoxic sensing and demethylation of nucleic acids and histones. Although substantial progress has been made in elucidating their function, the role of many 2OG dioxygenases remains enigmatic. Here we have studied the 2OG and iron (Fe(II)) dependent dioxygenase Ofd2 in Schizosaccharomyces pombe, a member of the AlkB subfamily of dioxygenases. We show that decarboxylation of 2OG by recombinant Ofd2 is dependent on Fe(II) and a histidine residue predicted to be involved in Fe(II) coordination. The decarboxylase activity of Ofd2 is stimulated by histones, and H2A has the strongest effect. Ofd2 interacts with all four core histones, however, only very weakly with H4. Our results define a new subclass of AlkB proteins interacting with histones, which also might comprise some of the human AlkB homologs with unknown function.

Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.

Genome-wide association analysis in primary sclerosing cholangitis identifies two non-HLA susceptibility loci.

Primary sclerosing cholangitis (PSC) is a chronic bile duct disease affecting 2.4-7.5% of individuals with inflammatory bowel disease. We performed a genome-wide association analysis of 2,466,182 SNPs in 715 individuals with PSC and 2,962 controls, followed by replication in 1,025 PSC cases and 2,174 controls. We detected non-HLA associations at rs3197999 in MST1 and rs6720394 near BCL2L11 (combined P = 1.1 × 10⁻¹6 and P = 4.1 × 10⁻8, respectively).

Studies on multiple forms of proGRP in serum from small cell lung cancer patients.

To determine whether various forms of progastrin-releasing peptide (proGRP) exist in the human circulation, a panel of monoclonal antibodies was produced to construct immunometric assays for various proGRP fragments. In combination with liquid chromatography, this facilitated the determination of fragment size distribution in the sera of patients with small cell lung cancer (SCLC). Separation of proGRP peptides from SCLC cell lines and patient samples by ion exchange and gel filtration resulted in the identification of several C-terminal fragments with wide differences in the relative amounts of each cleavage product. The high specificity of our immunometric assays for the various serum proGRP fragments indicates that they are promising tools for future investigations on the relationship between fragment distribution and diagnosis and prognosis.

Early-onset lymphoma and extensive embryonic apoptosis in two domain-specific Fen1 mice mutants.

Flap endonuclease 1 (FEN1) processes Okazaki fragments in lagging strand DNA synthesis, and FEN1 is involved in several DNA repair pathways. The interaction of FEN1 with the proliferating cell nuclear antigen (PCNA) processivity factor is central to the function of FEN1 in both DNA replication and repair. Here we present two gene-targeted mice with mutations in FEN1. The first mutant mouse carries a single amino acid point mutation in the active site of the nuclease domain of FEN1 (Fen1(E160D/E160D)), and the second mutant mouse contains two amino acid substitutions in the highly conserved PCNA interaction domain of FEN1 (Fen1(DeltaPCNA/DeltaPCNA)). Fen1(E160D/E160D) mice develop a considerably elevated incidence of B-cell lymphomas beginning at 6 months of age, particularly in females. By 16 months of age, more than 90% of the Fen1(E160D/E160D) females have tumors, primarily lymphomas. By contrast, Fen1(DeltaPCNA/DeltaPCNA) mouse embryos show extensive apoptosis in the forebrain and vertebrae area and die around stage E9.5 to E11.5.