Noncoding genetic variation is a major driver of phenotypic diversity, but functional interpretation is challenging. To better understand common genetic variation associated with brain diseases, we defined noncoding regulatory regions for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with variants in transcriptional enhancers and promoters in neurons, sporadic Alzheimer’s disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting disease-risk variants in cell-type–specific enhancers to promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated BIN1 expression in microglia, but not in neurons or astrocytes. These findings revise and expand the list of genes likely to be influenced by noncoding variants in AD and suggest the probable cell types in which they function.

Arrays of octameric peptide libraries on cellulose paper were screened by using 32 protein kinase inhibitor ͉ combinatorial libraries ͉ SPOT method ͉ membrane translocation signal ͉ smooth muscle T he cGMP-dependent protein kinases type I␣ and I␤ (cGPK) act directly downstream in the NO-mediated signaling pathway, controlling a variety of cellular responses, ranging from smooth muscle cell relaxation to neuronal synaptic plasticity (1, 2). The structural similarity of cGPK and its closest relative, the cAMP-dependent protein kinase (cAPK), has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily because of the lack of potent and selective cGPK inhibitors. Because recent studies have suggested that cAMP and cGMP are each able to cross-activate either cGPK or cAPK under physiological conditions, the specific role for cGPK within the NO͞cGMP-mediated signaling pathway remains obscure (for a review see ref. 1). However, recent advances have clearly identified specific intracellular targets for the cGPK isozymes I␣ and I␤ (3, 4). Also, inactivation of the genes for cGPK I␣͞I␤ and cGPK II showed that the cGPK isozymes regulate distinct cellular functions by pathways separate from those mediated by cAPK (5, 6).Attempts to identify cGPK-selective inhibitor peptides based on the autoinhibitory domain of the enzyme or in vivo substrates have been tedious at best, because of the lack of a well defined consensus sequence. Only a relative preference for basic residues surrounding the phosphate acceptor site has been established (7). Various synthetic peptides have been used to analyze the sequence requirements for cGPK substrates (8-11). Recently, we developed an iterative approach using phosphorylation of peptide libraries on cellulose paper to determine a priori the substrate specificity of cGPK versus cAPK. Consequently, we identified the cGPK substrate sequence TQAKRKKSLAM-FLR, in which the serine represents the phosphate-acceptor site (12, 13). Substitution of this serine by alanine yielded cGPK inhibitors with K i values of 7.5-22 M (13) and improved cGPK͞cAPK selectivity, as has been reported with other synthetic peptide derivatives (14,15). However, all cGPK peptide inhibitors known so far lack satisfactory potency and selectivity.Here we report a peptide library screen specifically designed to select for tight binding peptides rather than substrate peptides. First, we took advantage of the autophosphorylation properties of cGPK, which provides the means to study the transient enzyme-peptide interactions. Second, we used peptide libraries that lack the phosphate acceptor residues serine and threonine to select for peptide binding over phosphorylation. Linking the best sequence from this screen to membrane translocation signals (MTS) for intracellular delivery resulted in the highly effective cGPK I␣ inhibitors DT-2 and DT-3. Finally, we have demonstrated that both peptides are powerful tools for studying the specific functional roles of cGPK in smooth muscle. MethodsEnzyme Prepa...

The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here we report the 2.5 Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.

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