Yutaka Takaoka

Role of Intrapancreatic SPINK1/Spink3 Expression in the Development of Pancreatitis

Studies on hereditary pancreatitis have provided evidence in favor of central role for trypsin activity in the disease. Identification of genetic variants of trypsinogen linked the protease to the onset of pancreatitis, and biochemical characterization proposed an enzymatic gain of function as the initiating mechanism. Mutations of serine protease inhibitor Kazal type 1 gene (SPINK1) are shown to be associated with hereditary pancreatitis. We previously reported that Spink3 (a mouse homolog gene of human SPINK1) deficient mice showed excessive autophagy, followed by inappropriate trypsinogen activation in the exocrine pancreas. These data indicate that the role of SPINK1/Spink3 is not only trypsin inhibitor, but also negative regulator of autophagy. On the other hand, recent studies showed that high levels of SPINK1 protein detected in a serum or urine were associated with adverse outcome in various cancer types. It has been suggested that expression of SPINK1 and trypsin is balanced in normal tissue, but this balance could be disrupted during tumor progression. Based on the structural similarity between SPINK1 and epidermal growth factor (EGF), we showed that SPINK1 protein binds and activates EGF receptor, thus acting as a growth factor on tumor cell lines. In this review, we summarize the old and new roles of SPINK1/Spink3 in trypsin inhibition, autophagy, and cancer cell growth. These new functions of SPINK1/Spink3 may be related to the development of chronic pancreatitis.

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.

A polymorphic mutation, c.-3279T > G, in the UGT1A1 promoter is a risk factor for neonatal jaundice in the Malay population.

The uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene encodes the enzyme responsible for bilirubin glucuronidation. To evaluate the contribution of UGT1A1 promoter mutations to neonatal jaundice, we determined the genotypes of c.-3279T>G, c.-3156G>A, and A(TA)7TAA in Malay infants with neonatal jaundice (patients) and in infants without neonatal jaundice (controls). In our population study, only c.-3279T>G was associated with neonatal jaundice. The genotype distributions between both groups were significantly different (p = 0.003): the frequency of homozygosity for c.-3279G was much higher in patients than those in controls. Allele frequency of c.-3279G was significantly higher in patients than those in controls (p = 0.006). We then investigated changes in transcriptional activity because of c.-3279T>G. Luciferase reporter assay in HepG2 cells demonstrated that transcriptional activity of the c.-3279G allele was significantly lower than that of the c.-3279T allele in both the absence and presence of bilirubin. Luciferase reporter assay in COS-7 cells elucidated that c.-3279T>G modified the synergistic effects of the nuclear factors associated with transcriptional machinery. In conclusion, the c.-3279T>G mutation in the UGT1A1 promoter is a genetic risk factor for neonatal jaundice.

Two novel mutations in the ED1 gene in Japanese families with X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by hypodontia, hypotrichosis, and hypohidrosis, is caused by mutations in ED1, the gene encoding ectodysplasin-A (EDA). This protein belongs to the tumor necrosis factor ligand superfamily. We analyzed ED1 in two Japanese patients with XLHED. In patient 1, we identified a 4-nucleotide insertion, c.119-120insTGTG, in exon 1, which led to a frameshift mutation starting from that point (p.L40fsX100). The patients mother was heterozygous for this mutation. In patient 2, we identified a novel missense mutation, c.1141G>C, in exon 9, which led to a substitution of glycine with arginine in the TNFL domain of EDA (p.G381R). This patients mother and siblings showed neither symptoms nor ED1 mutations, so this mutation was believed to be a de novo mutation in maternal germline cells. According to molecular simulation analysis of protein structure and electrostatic surface, p.G381R increases the distance between K375 in monomer A and K327 in monomer B, which suggests an alteration of overall structure of EDA. Thus, we identified two novel mutations, p.L40fsX100 and p.G381R, in ED1 of two XLHED patients. Simulation analysis suggested that the p.G381R mutation hampers binding of EDA to its receptor via alteration of overall EDA structure.

Differences in Clinical Phenotype among Patients with XP Complementation Group D: 3D Structure and ATP-Docking of XPD In Silico

TO THE EDITOR Xeroderma pigmentosum (XP) is an autosomal recessive hereditary disease that is classified into seven genetic complementation groups, A through G, of nucleotide excision repair (NER)deficient types and one NER-proficient variant type (DiGiovanna and Kraemer, 2012). Patients with XP complementation group D (XPD) display photosensitivity, proneness to skin cancer and neurological symptoms, but the severity varies. In XPD from Western countries, the single amino-acid change R683W is found in 73% of the patients and most of them suffer from neurological symptoms (Takayama et al., 1995; Kobayashi et al., 1997; Taylor et al., 1997; Viprakasit et al., 2001; Kobayashi et al., 2002; Boyle et al., 2008; Emmert et al., 2009; Ueda et al., 2009). In contrast, Japanese patients display only skin manifestations without neurological symptoms (Kobayashi et al., 1997; Taylor et al., 1997; Kobayashi et al., 2002). XPD protein is an ATP-dependent 50-30 DNA helicase, which exerts unwinding of a damaged DNA strand to facilitate repair of the DNA. Although the human XPD protein remains refractory to crystallization, the crystal structure of XPD proteins from the archea has been solved (Fan et al., 2008; Liu et al., 2008). These structures revealed that XPD functions as a helicase with two helicase motifs separated by an ATP-binding cleft and two additional domains, a Fe–S cluster and an Arch domain. In this study, we present six cases of Japanese XPD without neurological abnormalities in four families. All patients showed severe photosensitivity since birth, and their skin in the sunexposed areas was hyperpigmented and covered with numerous pigmented maculae with color variations from light brown to dark brown. Four patients developed skin cancer, but none of them show neurological signs. XPD1KO and XPD4KO were assessed by a neurologist and an otorhinologist. Their clinical characteristics have been summarized in Table 1. To determine the ability to repair UV-induced DNA damage, a colonyformation assay after UV irradiation and UV-induced unscheduled DNA synthesis (UDS) was assessed (Materials and Methods are described in Supplementary data). The dose range giving 37% cell survival and UV-induced UDS in patients’ cells was much lower compared with that in the healthy subjects but was higher than that in XP-A cells (Table 1). Genetic complementation tests were carried out by means of a host-cell reactivation assay. Luciferase activity was increased specifically when XPD cDNA was cotransfected into each patient’s cells. Therefore, we concluded that all cases belonged to XPD. Direct sequence analysis was performed on each exon of the genomic DNA of Accepted article preview online 13 January 2014; published online 13 February 2014 Abbreviations: NER, nucleotide excision repair; UDS, unscheduled DNA synthesis; XP, xeroderma pigmentosum; XPD, xeroderma pigmentosum complementation group D E Nakano et al. Genotype–Phenotype and Protein Structure of XPD

Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers

BackgroundDuchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors.ResultsUsing a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group.ConclusionsThe decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.

Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits

BackgroundGlomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains.MethodsIn this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains.ResultsWe report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic.ConclusionsThis is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.

Hsp12.6 Expression Is Inducible by Host Immunity in Adult Worms of the Parasitic Nematode Nippostrongylus brasiliensis

Heat shock proteins (Hsp) are a family of stress-inducible molecular chaperones that play multiple roles in a wide variety of animals. However, the roles of Hsps in parasitic nematodes remain largely unknown. To elucidate the roles of Hsps in the survival and longevity of nematodes, particularly at the 2 most critical stages in their lifecycle, the infective-L3 stage and adult stage, which is subjected to host-derived immunological pressure, we examined the temporal gene transcription patterns of Hsp12.6, Hsp20, Hsp70, and Hsp90 throughout the developmental course of the nematode Nippostrongylus brasiliensis by reverse transcriptase real-time PCR. Nb-Hsp70 and Nb-Hsp90 expression were observed throughout the nematodes lifecycle, while the expression of Nb-Hsp20 was restricted to adults. Interestingly, Nb-Hsp12.6 showed a biphasic temporal expression pattern; i.e., it was expressed in infective-L3 larvae and in adults during worm expulsion from immunocompetent rats. However, the activation of Nb-Hsp12.6 in adult worms was aborted when they infected permissive athymic-rnu/rnu rats and was only marginal when they infected mast-cell-deficient Ws/Ws rats, which exhibited a low response of rat mast cell protease (RMCP) II and resistin-like molecule (Relm)- β expression compared to those observed in immunocompetent rats. Moreover, the activation of Nb-Hsp12.6 was reversed when adult worms were transplanted into the naive rat intestine. These features of Nb-Hsp12.6, the expression of which is not only stage-specific in infective-L3, but is also inducible by mucosal immunity in adults, have implications for the survival strategies of parasitic nematodes in deleterious environmental conditions both outside and inside the host.

CHF5074 (CSP-1103) stabilizes human transthyretin in mice humanized at the transthyretin and retinol-binding protein loci

Familial amyloidotic polyneuropathy is one type of protein misfolding disease. Transthyretin (TTR) tetramer dissociation is the limiting step for amyloid fibril formation. CHF5074 (CSP‐1103) stabilizes TTR tetramer in vitro by binding to the T4 binding site. Here, we used three strains of double humanized mice (mTtrhTTRVal30/hTTRVal30, mTtrhTTRVal30/hTTRMet30, and mTtrhTTRMet30/hTTRMet30) to assess whether CHF5074 stabilizes TTR tetramers in vivo. Treatment of mice with CHF5074 increased serum TTR levels by stabilizing TTR tetramers. Although the binding affinities of CHF5074 and diflunisal with TTRMet30 were similar, CHF5074 bound TTRVal30 more strongly than did diflunisal, suggesting the potent TTR‐stabilizing activity of CHF5074.

Ligand orientation governs conjugation capacity of UDP-glucuronosyltransferase 1A1.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is an endoplasmic reticulum membrane protein that catalyses glucuronidation. Mutant UGT1A1 possesses a different conjugation capacity, and the molecular mechanisms regulating these conjugation reactions are as yet unclear. To elucidate these molecular mechanisms, we simulated and analysed the glucuronidation of wild-type UGT1A1 and six UGT1A1 mutants, with bilirubin as the substrate. We found that only the orientation of the substrates correlated with the conjugation capacity in in vitro experiments. Inasmuch as glucuronidation is an intermolecular rearrangement reaction, we find that the conjugation reaction proceeds only when the hydroxyl group of the substrate is oriented towards the coenzyme, which allows the proton transfer to occur.