Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

STUDY QUESTION: Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER: Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY: Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION: This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS: Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE: PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION: Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS: Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S): Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

Characterization of a dengue NS4B inhibitor originating from an HCV small molecule library.

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.

BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.

BRAF mutation testing with a rapid, fully integrated molecular diagnostics system.

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTMBRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction-based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTMBRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation-detecting tests.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

Surface plasmon resonance as a tool to select potent drug candidates for hepatitis C virus NS5B polymerase.

Surface plasmon resonance (SPR)-based optical biosensors have been widely used to study biomolecular interactions, and applied to many areas of drug discovery including target identification, fragment screening, lead compound selection, early ADME (absorption, distribution, metabolism and excretion), and quality control. These biosensors allow the following of a biomolecular interaction in real time to monitor kinetics and determine affinity. In this chapter, we describe an SPR-based assay to measure the interaction between hepatitis C virus NS5B polymerase (wild type and/or mutants) and a small-molecule inhibitor. Viral polymerase proteins are captured on a Ni(2+)-nitrilotriacetic acid sensor surface while the small--molecule inhibitors are passed over the surface. In this way kinetics and affinity of the enzyme-inhibitor interactions can be measured, making it possible to select potent and promising lead candidates.

Expression and purification of dengue virus NS5 polymerase and development of a high-throughput enzymatic assay for screening inhibitors of dengue polymerase.

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 µM) and promising pharmacokinetic profiles in rats and dogs. TMC647055 is currently being evaluated in the clinic.

Polycystin-2 activation by inositol 1,4,5-trisphosphate-induced Ca2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling microdomain.

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Unraveling the role of polycystin-2/inositol 1,4,5-trisphosphate receptor interaction in Ca signaling.

Autosomal dominant polycystic kidney disease (ADPKD) arises as a consequence of mutations of the genes PKD1 and PKD2, encoding respectively the integral membrane proteins polycystin-1 and polycystin-2 (TRPP2), resulting in a disturbance in intracellular Ca(2+) signaling. Previously we investigated the interaction between TRPP2 and the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum (ER). We identified the molecular determinants of this interaction and observed an enhanced IP(3)-induced Ca(2+) release (IICR). Since we found that TRPP2 strongly bound to a cluster of positively charged amino acids in the N-terminal ligand-binding domain (LBD) of the IP(3)R, we now investigated whether TRPP2 would interfere with the binding of IP(3) to the IP(3)R. In in vitro experiments we observed that TRPP2 partially inhibited the binding of IP(3) to the LBD of the IP(3)R with an IC(50) of ∼350 nM. The suppressor domain, i.e., the N-terminal 225 amino acids of the LBD of the IP(3)R, mediated this inhibitory effect of TRPP2 on IP(3) binding. The observation that the interaction between the IP(3)R and TRPP2 decreased IP(3) binding is in apparent contrast to the increased IICR. The data can be explained however by a subsequent activation of Ca(2+)-induced Ca(2+) release (CICR) via TRPP2. Implications of this mechanism for cellular Ca(2+) signaling are discussed in this addendum.

Structure-based design of a benzodiazepine scaffold yields a potent allosteric inhibitor of hepatitis C NS5B RNA polymerase.

HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

Heterogeneous nuclear ribonucleoprotein h1, a novel nuclear autoantigen.

BACKGROUND: Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (>or=640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified. METHODS: Using an immunoproteomics approach we identified heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) as a novel nuclear target of autoantibody response. RESULTS: Recombinant rat hnRNP H1 reacted in Western blot analyses with 48% of 93 sera from patients with primary Sjögren syndrome and with 5.2% of 153 sera from patients with other connective tissue diseases (diseased controls). For comparison, the diagnostic sensitivity and specificity of anti-Sjögren syndrome A (SSA) antibodies for primary Sjögren syndrome in the same patient cohort were 88.2% and 76.3%, respectively. Interestingly, 5 of 11 primary Sjögren syndrome patients with no anti-SSA or anti-SSB antibodies had anti-hnRNP H1 antibodies. Anti-hnRNP H1 antibodies were preabsorbed by hnRNP H1, as demonstrated by indirect immunofluorescence. In an evaluation of the presence of anti-hnRNP H1 antibodies in 188 consecutive samples submitted to the clinical laboratory with positive ANA (titer >or=160), anti-hnRNP H1 antibodies were found in 3 of 7 (2 primary and 5 secondary) Sjögren syndrome patients and in 8.3% of the diseased controls. CONCLUSIONS: HnRNP H1 is a newly discovered autoantigen that could become an additional diagnostic marker.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a universal intracellular Ca2+-release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP3R or with associated proteins and so provide a large potential for fine tuning the Ca2+-release activity and for creating efficient Ca2+ signals in subcellular microdomains.

The IRBIT domain adds new functions to the AHCY family.

During the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) intracellular Ca2+ via the inositol 1,4,5-trisphosphate receptor (IP3R), (ii) intracellular pH via the Na+/HCO3 - cotransporters (NBCs); whereas inactivation of the IRBIT domain induces (iii) nuclear translocation and regulation of AHCY activity. Dysfunction of AHCY-like proteins will disturb these three important functions, with various biological implications.

The complex regulatory function of the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) can be divided in three functionally distinct regions: a ligand-binding domain, a modulatory domain and a channel domain. Numerous regulatory mechanisms including inter- and intra-molecular protein-protein interactions and phosphorylation events act via these domains to regulate the function of the IP(3)R. Regulation at the level of the ligand-binding domain primarily affects the affinity for IP(3). The extent of IP(3)-induced Ca(2+) release (IICR) is, however, not only determined by the affinity for IP(3) but also by the effectiveness of the coupling between ligand binding and channel opening. As a result, regulation as well as malfunction of IICR may be affected by both steps in the activation mechanism. The 3D structures of the two subdomains of the ligand-binding domain have recently been determined by X-ray diffraction analysis. This allows a more detailed molecular explanation of the regulatory events situated at the ligand-binding domain of the IP(3)R. In this review, we will focus on recent structural and functional data on the ligand-binding domain that have extended and clarified the view on the molecular mechanisms of IP(3)R regulation.

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor.

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.

Molecular and functional characterization of inositol trisphosphate receptors during early zebrafish development.

Fluctuations in cytosolic Ca(2+) are crucial for a variety of cellular processes including many aspects of development. Mobilization of intracellular Ca(2+) stores via the production of inositol trisphosphate (IP(3)) and the consequent activation of IP(3)-sensitive Ca(2+) channels is a ubiquitous means by which diverse stimuli mediate their cellular effects. Although IP(3) receptors have been well studied at fertilization, information regarding their possible involvement during subsequent development is scant. In the present study we examined the role of IP(3) receptors in early development of the zebrafish. We report the first molecular analysis of zebrafish IP(3) receptors which indicates that, like mammals, the zebrafish genome contains three distinct IP(3) receptor genes. mRNA for all isoforms was detectable at differing levels by the 64 cell stage, and IP(3)-induced Ca(2+) transients could be readily generated (by flash photolysis) in a controlled fashion throughout the cleavage period in vivo. Furthermore, we show that early blastula formation was disrupted by pharmacological blockade of IP(3) receptors or phospholipase C, by molecular inhibition of the former by injection of IRBIT (IP(3) receptor-binding protein released with IP(3)) and by depletion of thapsigargin-sensitive Ca(2+) stores after completion of the second cell cycle. Inhibition of Ca(2+) entry or ryanodine receptors, however, had little effect. Our work defines the importance of IP(3) receptors during early development of a genetically and optically tractable model vertebrate organism.