With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting β-can domains was compared with that of flexible linkers or of a three α-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2–5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an α-helix and the α-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This `linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.

Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was Fabp7 (fatty acid binding protein 7, brain), a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTL's proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming. A startle response to an unexpected, strong startling stimulus can be suppressed by an immediately preceding low-intensity stimulus, thereby eliciting little behavioral response. This phenomenon, called prepulse inhibition (PPI), has been observed in all mammals tested and is thought to reflect sensory-motor gating functions in organisms. PPI is diminished in human schizophrenia, raising the possibility that PPI might serve as a potential biological marker for the disease. Once the genes regulating PPI in lower animals are identified, it is expected that the human orthologs will be strong candidate genes for schizophrenia. In this study, we first performed a genetic dissection of mouse PPI using quantitative trait loci analysis, which detects chromosomal regions harboring causative genes. Further analyses including those of knockout mice, allowed us to identify one potential causative gene, Fabp7 (fatty acid binding protein 7, brain), a chaperon for the essential fatty acid docosahexaenoic acid. Human studies showed that the FABP7 gene is modestly associated with schizophrenia and that transcript expression levels are up-regulated in schizophrenic brains. From these results, we propose that a FABP7 protein-mediated disturbance of essential lipid metabolism in developing brains may be one risk factor in the development of schizophrenia, with a greater effect in males.

Disturbances of lipid metabolism have been implicated in psychiatric illnesses. We previously reported an association between the gene for fatty acid binding protein 7 (FABP7) and schizophrenia. Furthermore, we identified and reported several rare non-synonymous polymorphisms of the brain-expressed genes FABP3, FABP5 and FABP7 from schizophrenia and autism spectrum disorder (ASD), diseases known to part share genetic architecture. Here, we conducted further studies to better understand the contribution these genes make to the pathogenesis of schizophrenia and ASD. In postmortem brains, we detected altered mRNA expression levels of FABP5 in schizophrenia, and of FABP7 in ASD and altered FABP5 in peripheral lymphocytes. Using a patient cohort, comprehensive mutation screening identified six missense and two frameshift variants from the three FABP genes. The two frameshift proteins, FABP3 E132fs and FABP7 N80fs, formed cellular aggregates and were unstable when expressed in cultured cells. The four missense mutants with predicted possible damaging outcomes showed no changes in intracellular localization. Examining ligand binding properties, FABP7 S86G and FABP7 V126L lost their preference for docosahexaenoic acid to linoleic acid. Finally, mice deficient in Fabp3, Fabp5 and Fabp7 were evaluated in a systematic behavioral test battery. The Fabp3 knockout (KO) mice showed decreased social memory and novelty seeking, and Fabp7 KO mice displayed hyperactive and anxiety-related phenotypes, while Fabp5 KO mice showed no apparent phenotypes. In conclusion, disturbances in brain-expressed FABPs could represent an underlying disease mechanism in a proportion of schizophrenia and ASD sufferers.