High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Network Clustering Revealed the Systemic Alterations of Mitochondrial Protein Expression

The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ0) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ0 cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions

FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study.

Contains fulltext : 95893.pdf (publisher's version ) (Closed access)BACKGROUND: Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS: We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS: We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132.0 (95% CI 38.7-450.3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92.3% (95% CI 81.5-97.9%) and specificity was 91.7% (84.8-96.1%). The positive and negative predictive values for FGF-21 were 84.2% (95% CI 72.1-92.5%) and 96.1 (90.4-98.9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0.95; by comparison, the values for other biomarkers were 0.83 lactate (p=0.037, 0.83 for pyruvate (p=0.015), 0.72 for the lactate-to-pyruvate ratio (p=0.0002), and 0.77 for creatine kinase (p=0.013). INTERPRETATION: Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING: Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppo Foundation.1 september 201

Altered physiological brain variation in drug‐resistant epilepsy

Abstract Introduction: Functional magnetic resonance imaging (fMRI) combined with simultaneous electroencephalography (EEG‐fMRI) has become a major tool in mapping epilepsy sources. In the absence of detectable epileptiform activity, the resting state fMRI may still detect changes in the blood oxygen level‐dependent signal, suggesting intrinsic alterations in the underlying brain physiology. Methods: In this study, we used coefficient of variation (CV) of critically sampled 10 Hz ultra‐fast fMRI (magnetoencephalography, MREG) signal to compare physiological variance between healthy controls (n = 10) and patients (n = 10) with drug‐resistant epilepsy (DRE). Results: We showed highly significant voxel‐level (p < 0.01, TFCE‐corrected) increase in the physiological variance in DRE patients. At individual level, the elevations range over three standard deviations (σ) above the control mean (μ) CVMREG values solely in DRE patients, enabling patient‐specific mapping of elevated physiological variance. The most apparent differences in group‐level analysis are found on white matter, brainstem, and cerebellum. Respiratory (0.12–0.4 Hz) and very‐low‐frequency (VLF = 0.009–0.1 Hz) signal variances were most affected. Conclusions: The CVMREG increase was not explained by head motion or physiological cardiorespiratory activity, that is, it seems to be linked to intrinsic physiological pulsations. We suggest that intrinsic brain pulsations play a role in DRE and that critically sampled fMRI may provide a powerful tool for their identification