Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 µM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

Quantifying signaling pathway activation to monitor the quality of induced pluripotent stem cells.

Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict in vitro differentiation potential. Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applications.

Regulation of hippocampal synaptic plasticity thresholds and changes in exploratory and learning behavior in dominant negative NPR-B mutant rats.

The second messenger cyclic GMP affects synaptic transmission and modulates synaptic plasticity and certain types of learning and memory processes. The impact of the natriuretic peptide receptor B (NPR-B) and its ligand C-type natriuretic peptide (CNP), one of several cGMP producing signaling systems, on hippocampal synaptic plasticity and learning is, however, less well understood. We have previously shown that the NPR-B ligand CNP increases the magnitude of long-term depression (LTD) in hippocampal area CA1, while reducing the induction of long-term potentiation (LTP). We have extended this line of research to show that bidirectional plasticity is affected in the opposite way in rats expressing a dominant-negative mutant of NPR-B (NSE-NPR-BΔKC) lacking the intracellular guanylyl cyclase domain under control of a promoter for neuron-specific enolase. The brain cells of these transgenic rats express functional dimers of the NPR-B receptor containing the dominant-negative NPR-BΔKC mutant, and therefore show decreased CNP-stimulated cGMP-production in brain membranes. The NPR-B transgenic rats display enhanced LTP but reduced LTD in hippocampal slices. When the frequency-dependence of synaptic modification to afferent stimulation in the range of 1-100 Hz was assessed in transgenic rats, the threshold for both, LTP and LTD induction, was shifted to lower frequencies. In parallel, NPR-BΔKC rats exhibited an enhancement in exploratory and learning behavior. These results indicate that bidirectional plasticity and learning and memory mechanism are affected in transgenic rats expressing a dominant-negative mutant of NPR-B. Our data substantiate the hypothesis that NPR-B-dependent cGMP signaling has a modulatory role for synaptic information storage and learning.

The putative tumor suppressor VILIP-1 counteracts epidermal growth factor-induced epidermal-mesenchymal transition in squamous carcinoma cells.

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.

The visinin-like proteins VILIP-1 and VILIP-3 in Alzheimer's disease-old wine in new bottles.

The neuronal Ca(2+)-sensor (NCS) proteins VILIP-1 and VILIP-3 have been implicated in the etiology of Alzheimer's disease (AD). Genome-wide association studies (GWAS) show association of genetic variants of VILIP-1 (VSNL1) and VILIP-3 (HPCAL1) with AD+P (+psychosis) and late onset AD (LOAD), respectively. In AD brains the expression of VILIP-1 and VILIP-3 protein and mRNA is down-regulated in cortical and limbic areas. In the hippocampus, for instance, reduced VILIP-1 mRNA levels correlate with the content of neurofibrillary tangles (NFT) and amyloid plaques, the pathological characteristics of AD, and with the mini mental state exam (MMSE), a test for cognitive impairment. More recently, VILIP-1 was evaluated as a cerebrospinal fluid (CSF) biomarker and a prognostic marker for cognitive decline in AD. In CSF increased VILIP-1 levels correlate with levels of Aß, tau, ApoE4, and reduced MMSE scores. These findings tie in with previous results showing that VILIP-1 is involved in pathological mechanisms of altered Ca(2+)-homeostasis leading to neuronal loss. In PC12 cells, depending on co-expression with the neuroprotective Ca(2+)-buffer calbindin D28K, VILIP-1 enhanced tau phosphorylation and cell death. On the other hand, VILIP-1 affects processes, such as cyclic nucleotide signaling and dendritic growth, as well as nicotinergic modulation of neuronal network activity, both of which regulate synaptic plasticity and cognition. Similar to VILIP-1, its interaction partner α4ß2 nicotinic acetylcholine receptor (nAChR) is severely reduced in AD, causing severe cognitive deficits. Comparatively little is known about VILIP-3, but its interaction with cytochrome b5, which is part of an antioxidative system impaired in AD, hint toward a role in neuroprotection. A current hypothesis is that the reduced expression of visinin-like protein (VSNLs) in AD is caused by selective vulnerability of subpopulations of neurons, leading to the death of these VILIP-1-expressing neurons, explaining its increased CSF levels. While the Ca(2+)-sensor appears to be a good biomarker for the detrimental effects of Aß in AD, its early, possibly Aß-induced, down-regulation of expression may additionally attenuate neuronal signal pathways regulating the functions of dendrites and neuroplasticity, and as a consequence, this may contribute to cognitive decline in early AD.

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1).

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 µM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 µM and K(EF2) = 1-4 µM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 µM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.