Expanding the diversity of chemical protein modification allows post-translational mimicry

One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. We therefore anticipate that this ability to build model systems will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein's PTMs from an in vivo assay into an in vitro system.     

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.     

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.     

Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins

During protein biosynthesis, nascent polypeptide chains that emerge from the ribosomal exit tunnel encounter ribosome-associated chaperones, which assist their folding to the native state. Here we present a 2.7 A crystal structure of Escherichia coli trigger factor, the best-characterized chaperone of this type, together with the structure of its ribosome-binding domain in complex with the Haloarcula marismortui large ribosomal subunit. Trigger factor adopts a unique conformation resembling a crouching dragon with separated domains forming the amino-terminal ribosome-binding 'tail', the peptidyl-prolyl isomerase 'head', the carboxy-terminal 'arms' and connecting regions building up the 'back'. From its attachment point on the ribosome, trigger factor projects the extended domains over the exit of the ribosomal tunnel, creating a protected folding space where nascent polypeptides may be shielded from proteases and aggregation. This study sheds new light on our understanding of co-translational protein folding, and suggests an unexpected mechanism of action for ribosome-associated chaperones.     

Multiplicative computation in a visual neuron sensitive to looming

Multiplicative operations are important in sensory processing, but their biophysical implementation remains largely unknown. We investigated an identified neuron (the lobula giant movement detector, LGMD, of locusts) whose output firing rate in response to looming visual stimuli has been described by two models, one of which involves a multiplication. In this model, the LGMD multiplies postsynaptically two inputs (one excitatory, one inhibitory) that converge onto its dendritic tree; in the other model, inhibition is presynaptic to the LGMD. By using selective activation and inactivation of pre- and postsynaptic inhibition, we show that postsynaptic inhibition has a predominant role, suggesting that multiplication is implemented within the neuron itself. Our pharmacological experiments and measurements of firing rate versus membrane potential also reveal that sodium channels act both to advance the response of the LGMD in time and to map membrane potential to firing rate in a nearly exponential manner. These results are consistent with an implementation of multiplication based on dendritic subtraction of two converging inputs encoded logarithmically, followed by exponentiation through active membrane conductances.     

Systematic screen for human disease genes in yeast

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.     

miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration

microRNAs (miRNAs) function as genetic rheostats to control gene output. Based on their role as modulators, it has been postulated that miRNAs canalize development and provide genetic robustness. Here, we uncover a previously unidentified regulatory layer of chemokine signaling by miRNAs that confers genetic robustness on primordial germ cell (PGC) migration. In zebrafish, PGCs are guided to the gonad by the ligand Sdf1a, which is regulated by the sequestration receptor Cxcr7b. We find that miR-430 regulates sdf1a and cxcr7 mRNAs. Using target protectors, we demonstrate that miR-430-mediated regulation of endogenous sdf1a (also known as cxcl12a) and cxcr7b (i) facilitates dynamic expression of sdf1a by clearing its mRNA from previous expression domains, (ii) modulates the levels of the decoy receptor Cxcr7b to avoid excessive depletion of Sdf1a and (iii) buffers against variation in gene dosage of chemokine signaling components to ensure accurate PGC migration. Our results indicate that losing miRNA-mediated regulation can expose otherwise buffered genetic lesions leading to developmental defects.     

NATREX AND DETERMINATION OF REAL EXCHANGE RATE OF RMB

1 IntroductionIn the past decades, a lot of 1iteratures appeared in the determination of excfiange rate, boththeoretic and empirical. One preValing paradigm is that the puxchasing power parity (PPP)holds in the long run either in weak--fOrm or in strongform. Under this hyPothesis, fluctuationsin real exchange rates are often regaJrded as temporary deviations from the long--run equllibriumexchange rate. However, these ekisting models fail to eXPlain the 1arge fluctuations in the realexchang…     

SLC2A9 influences uric acid concentrations with pronounced sex-specific effects

Serum uric acid concentrations are correlated with gout and clinical entities such as cardiovascular disease and diabetes. In the genome-wide association study KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K (n = 1,644), the most significant SNPs associated with uric acid concentrations mapped within introns 4 and 6 of SLC2A9, a gene encoding a putative hexose transporter (effects: -0.23 to -0.36 mg/dl per copy of the minor allele). We replicated these findings in three independent samples from Germany (KORA S4 and SHIP (Study of Health in Pomerania)) and Austria (SAPHIR; Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk), with P values ranging from 1.2 x 10(-8) to 1.0 x 10(-32). Analysis of whole blood RNA expression profiles from a KORA F3 500K subgroup (n = 117) showed a significant association between the SLC2A9 isoform 2 and urate concentrations. The SLC2A9 genotypes also showed significant association with self-reported gout. The proportion of the variance of serum uric acid concentrations explained by genotypes was about 1.2% in men and 6% in women, and the percentage accounted for by expression levels was 3.5% in men and 15% in women.     

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-β. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β are strongly associated with Alzheimer's disease, are more toxic than amyloid-β, residues 1-42 (Aβ(1-42)) and Aβ(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aβ may trigger Alzheimer's disease. Aβ(3(pE)-42) co-oligomerizes with excess Aβ(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ(3(pE)-42) plus 95% Aβ(1-42) (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ(1-42) monomers in the absence of additional Aβ(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aβ(3(pE)-42), and enhanced Aβ(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aβ(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.