Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words

Binding profile of Pin1-binding sites 1 and 2 in BNIP-H.

<p>The binding between different forms of BNIP-H and Pin1 are as detailed in the text. The + sign denotes relative strength of interactions as determined by co-immunoprecipitation studies. BNIP-H aa 1–287 harbors binding site 1 (aa 191–206) and BNIP-H Δ aa 189–287 contains binding site 2 (aa 287–332).</p

BNIP-H contains two Pin1-binding sites and interacts with the WW domain of Pin1.

<p>Purified GST fusion proteins of Pin1 full length, Pin1 WW domain and Pin1 PPI domain were incubated with PC12 whole cell lysates containing either HA-BNIP-H full length or various BNIP-H deletions proteins, which were expressed under NGF-stimulation (A, <i>in vitro</i> binding). After incubation, GST fusion proteins were isolated, washed and analyzed for the presence of bound BNIP-H or its mutants by western-blotting with anti-HA antibody. The membrane with the blotted proteins was than stained with amido black to reveal equal loading of GST and GST fusion proteins. GST protein was used as a control. Input shows approximately 2% of the lysate used for the GST pull-down assay. I, input; G, GST; P, Pin1 full length; WW, WW domain of Pin1; PPI, PPI domain of Pin1 (B). Various HA-tagged BNIP-H expression constructs (A, <i>in vivo</i> binding) and a FLAG-Pin1 expression plasmid were co-transfected into 293T cells. Lysates were subjected to immunoprecipitation with anti-FLAG antibody. Precipitates and whole cell lysates were analyzed by western-blotting with the indicated antibodies to detect binding of the different HA-BNIP-H fragments. A summary of the results is presented (A, <i>in vivo</i> binding) (C). 293T cells were transfected with expression plasmids for FLAG-BNIP-H full length, FLAG-BNIP-H aa 1–287, FLAG-BNIP-H Δ aa 189–287 or left untransfected. Lysates were subjected to immunoprecipitation with anti-FLAG antibody conjugated to agarose beads, and precipitates were washed thoroughly with RIPA buffer and RIPA buffer supplemented with 0.1–0.5% sodium dodecyl sulfate or 500 mM NaCl as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002686#s4" target="_blank">Material and Methods</a>”. Precipitates were resuspended in lysis buffer and incubated with recombinant Pin1 expressed and purified from <i>E.coli</i>. After sedimentation and washing, precipitates were analyzed by western-blotting with anti-Pin1 antibody (lanes 6–9). Lane 1 shows 10% of the input for recombinant Pin1. Lanes 2–5 demonstrate that the purified FLAG-BNIP-H constructs were devoid of endogenous Pin1 from 293T cells. The amido black-stained membrane shows equal input for the FLAG-BNIP-H constructs.</p

Pin1 competes with glutaminase for binding to BNIP-H in PC12 cells.

<p>PC12 cells were transfected with an expression plasmid for FLAG-BNIP-H full length alone or together with constructs for HA-Pin1 or HA-Rac1 as indicated. Cells were treated with NGF for 24 hours. After immunoprecipitation with anti-FLAG antibody, samples were analyzed by western-blotting with the indicated antibodies to reveal binding of endogenous kidney-type glutaminase to BNIP-H in the absence (lane 1) or presence of Pin1 (lane 2) or Rac1(lane 3). Samples shown were from the same experiment and analyzed on a single blot.</p

The two Pin1-binding sites within BNIP-H have different binding profiles.

<p>293T cells were transfected with expression plasmids for HA-BNIP-H aa 1–287 (A) or HA-BNIP-H Δ aa 189–287 (B) alone or together with expression plasmids for full length FLAG-tagged Pin1 wt, different full length FLAG-tagged point mutants or expression plasmids for FLAG-tagged PPI domain of Pin1 with or without the point mutation C113A. After immunoprecipitation with anti-FLAG antibody, samples were analyzed by western-blotting with the indicated antibodies to reveal binding of the two different HA-tagged BNIP-H fragments.</p

Intramolecular binding in BNIP-H.

<p>293T cells were transfected with expression plasmids for HA-BNIP-H full length, HA-BNIP-H aa 1–287 or HA-BNIP-H aa 1–190 alone or together with an expression construct for a FLAG-tagged C-terminal BNIP-H fragment (aa 286–371). A schematic picture of these fragments is shown (A). After co-immunoprecipitation with anti-FLAG antibody samples were analyzed by western-blotting with the indicated antibodies to reveal binding of the different HA-tagged fragments (B).</p

BNIP-H co-localizes with Pin1 in differentiating PC12 and P19 cells.

<p>PC12 (A) and P19 cells (B) were fixed, permeabilized and probed with the indicated antibodies, followed by appropriate fluorophore-conjugated secondary antibodies and analyzed by confocal microscopy. PC12 cells were treated with NGF for 24 and 48 hours, respectively (panel I). Undifferentiated PC12 cells grown for 48 hours were used in panel II. P19 cells were treated with retinoic acid for five days and allowed to differentiate for another six days without retinoic acid. Scale bar in (A) 20 µm for panel I, and 10 µm for panel II, in (B) 10 µm.</p

The effect of genotype and in utero environment on interindividual variation in neonate DNA methylomes

Integrating the genotype with epigenetic marks holds the promise of better understanding the biology that underlies the complex interactions of inherited and environmental components that define the developmental origins of a range of disorders. The quality of the in utero environment significantly influences health over the lifecourse. Epigenetics, and in particular DNA methylation marks, have been postulated as a mechanism for the enduring effects of the prenatal environment. Accordingly, neonate methylomes contain molecular memory of the individual in utero experience. However, interindividual variation in methylation can also be a consequence of DNA sequence polymorphisms that result in methylation quantitative trait loci (methQTLs) and, potentially, the interaction between fixed genetic variation and environmental influences. We surveyed the genotypes and DNA methylomes of 237 neonates and found 1423 punctuate regions of the methylome that were highly variable across individuals, termed variably methylated regions (VMRs), against a backdrop of homogeneity. MethQTLs were readily detected in neonatal methylomes, and genotype alone best explained ?25% of the VMRs. We found that the best explanation for 75% of VMRs was the interaction of genotype with different in utero environments, including maternal smoking, maternal depression, maternal BMI, infant birth weight, gestational age, and birth order. Our study sheds new light on the complex relationship between biological inheritance as represented by genotype and individual prenatal experience and suggests the importance of considering both fixed genetic variation and environmental factors in interpreting epigenetic variation

SRSF9 selectively represses ADAR2-mediated editing of brain-specific sites in primates

Adenosine-to-inosine (A-to-I) RNA editing displays diverse spatial patterns across different tissues. However, the human genome encodes only two catalytically active editing enzymes (ADAR1 and ADAR2), suggesting that other regulatory factors help shape the editing landscape. Here, we show that the splicing factor SRSF9 selectively controls the editing of many brain-specific sites in primates. SRSF9 is more lowly expressed in the brain than in non-brain tissues. Gene perturbation experiments and minigene analysis of candidate sites demonstrated that SRSF9 could robustly repress A-to-I editing by ADAR2. We found that SRSF9 biochemically interacted with ADAR2 in the nucleus via its RRM2 domain. This interaction required the presence of the RNA substrate and disrupted the formation of ADAR2 dimers. Transcriptome-wide location analysis and RNA sequencing revealed 1328 editing sites that are controlled directly by SRSF9. This regulon is significantly enriched for brain-specific sites. We further uncovered a novel motif in the ADAR2-dependent SRSF9 binding sites and provided evidence that the splicing factor prevents loss of cell viability by inhibiting ADAR2-mediated editing of genes involved in proteostasis, energy metabolism, the cell cycle and DNA repair. Collectively, our results highlight the importance of SRSF9 as an editing regulator and suggest potential roles for other splicing factors.ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio