Fiona Whelan

Phosphodiesterase genes are associated with susceptibility to major depression and antidepressant treatment response

Cyclic nucleotide phosphodiesterases (PDEs) constitute a family of enzymes that degrade cAMP and cGMP. Intracellular cyclic nucleotide levels increase in response to extracellular stimulation by hormones, neurotransmitters, or growth factors and are down-regulated through hydrolysis catalyzed by PDEs, which are therefore candidate therapeutic targets. cAMP is a second messenger implicated in learning, memory, and mood, and cGMP modulates nervous system processes that are controlled by the nitric oxide (NO)/cGMP pathway. To investigate an association between genes encoding PDEs and susceptibility to major depressive disorder (MDD), we genotyped SNPs in 21 genes of this superfamily in 284 depressed Mexican Americans who participated in a prospective, double-blind, pharmacogenetic study of antidepressant response, and 331 matched controls. Polymorphisms in PDE9A and PDE11A were found to be associated with the diagnosis of MDD. Our data are also suggestive of the association between SNPs in other PDE genes and MDD. Remission on antidepressants was significantly associated with polymorphisms in PDE1A and PDE11A. Thus, we found significant associations with both the diagnosis of MDD and remission in response to antidepressants with SNPs in the PDE11A gene. We show here that PDE11A haplotype GAACC is significantly associated with MDD. We conclude that PDE11A has a role in the pathophysiology of MDD. This study identifies a potential CNS role for the PDE11 family. The hypothesis that drugs affecting PDE function, particularly cGMP-related PDEs, represent a treatment strategy for major depression should therefore be tested.

The brain-derived neurotrophic factor rs6265 (Val66Met) polymorphism and depression in Mexican-Americans

The hypothesis that brain-derived neurotrophic factor (BDNF) is involved in the pathogenesis of major depression is supported by several research findings; however, genetic studies assessing the relationship between BDNF and psychiatric disorders have produced conflicting results. We examined the effect of a BDNF polymorphism on depression susceptibility in Mexican-Americans. The single nucleotide polymorphism (Val66Met), which has been shown to have functional and behavioral effects, was genotyped in 284 depressed participants and 331 controls, showing association with depression (P=0.005). Individuals homozygous for the major allele (GG) had an increased chance of being depressed (OR=1.7 95% CI 1.17–2.47). Our findings support the association of BDNF single nucleotide polymorphism rs6265 and depression, suggesting that this polymorphism may increase susceptibility to major depression in Mexican-Americans.

The pleiotropy of dioxin toxicity — Xenobiotic misappropriation of the aryl hydrocarbon receptor's alternative physiological roles

The aryl hydrocarbon receptor is a signal regulated transcription factor that has best been characterised as regulating the xenobiotic response to a variety of planar aromatic hydrocarbons. There is compelling evidence that it mediates most, if not all, of the toxic effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Dioxin exposure results in a wide variety of toxic outcomes including severe wasting syndrome, chloracne, thymic involution, severe immune suppression, reduced fertility, hepatotoxicity, teratogenicity, tumour promotion and death. The pleiotropy of toxic outcomes implies the disruption of a wide range of normal physiological functions. The aryl hydrocarbon receptor has developmentally restricted expression as well as developmental defects in gene-targeted mice. It has a wide range of target genes that do not fit into the classical xenobiotic metabolising gene battery and has recently been shown to interact with NF-kappa B and the estrogen receptor. There is also evidence for its activation in the absence of exogenous ligand, all of which point to various roles outside xenobiotic metabolism. Ligands so far identified display differential activation potential with respect to receptor activity. This article addresses activities of the aryl hydrocarbon receptor that are outside the xenobiotic response. Known physiological roles are discussed as well as how their disruption contributes to the pleiotropic toxicity of TCDD.

Caspase 1 deficiency reduces inflammation-induced brain transcription

The systemic inflammatory response syndrome (SIRS) is a life-threatening medical condition characterized by a severe and generalized inflammatory state that can lead to multiple organ failure and shock. The CNS regulates many features of SIRS such as fever, cardiovascular, and neuroendocrine responses. Central and systemic manifestations of SIRS can be induced by LPS or IL-1β administration. The crucial role of IL-1β in inflammation has been further highlighted by studies of mice lacking caspase 1 (casp1, also known as IL-1β convertase), a protease that cleaves pro-IL-1β into mature IL-1β. Indeed, casp1 knockout (casp1−/−) mice survive lethal doses of LPS. The key role of IL-1β in sickness behavior and its de novo expression in the CNS during inflammation led us to test the hypothesis that IL-1β plays a major role modulating the brain transcriptome during SIRS. We show a gene–environment effect caused by LPS administration in casp1−/− mice. During SIRS, the expression of several genes, such as chemokines, GTPases, the metalloprotease ADAMTS1, IL-1RA, the inducible nitric oxide synthase, and cyclooxygenase-2, was differentially increased in casp1−/− mice. Our findings may contribute to the understanding of the molecular changes that take place within the CNS during sepsis and SIRS and the development of new therapies for these serious conditions. Our results indicate that those genes may also play a role in several neuropsychiatric conditions in which inflammation has been implicated and indicate that casp1 might be a potential therapeutic target for such disorders.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

Significance RNA-binding proteins use diverse mechanisms for generating specificity toward distinct RNA molecules. Different subfamilies of bacterial dihydrouridine synthases (Dus) modify specific uridines in tRNA, but the mechanism for selection of the target nucleotide is unknown. We solved crystal structures of the U16-specific Dus from Escherichia coli complexed with two different tRNAs. These structures reveal that the tRNA is bound in a completely different orientation from that observed in a U20-specific enzyme. The major reorientation of the substrate tRNA, driven by unique amino acid “binding signatures” and plasticity in the position of the C-terminal recognition domain, appears to be an evolutionary innovation to the known strategies that define specificity of enzymes toward tRNA. The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding

The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit–subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible ‘brace’ maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

Sulfonation and Phosphorylation of Regions of the Dioxin Receptor Susceptible to Methionine Modifications

Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu409–Arg424 and Ser547–Arg555 of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H216O and H218O, and enzymatic dephosphorylation was subsequently performed on the H216O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu409–Arg424. This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group.

Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS

AbstractCollision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstractᅟ

Disorder drives cooperative folding in a multidomain protein

Significance Understanding the role played by disorder in biology is becoming increasingly important. Disordered proteins are central to signaling, development, initiation of transcription, and other vital cellular processes. How and why disordered proteins are used is not entirely clear, but disorder can be important in allostery, facilitate regulatory posttranslational modification, and allow rapid and specific but promiscuous binding. Here, our investigations of biofilm-promoting protein SasG illustrate that disorder can play another role. We show that the intrinsic disorder of one-half of the domains is important for imparting long-range cooperativity in folding of a large multidomain protein—allowing formation of a small local element of structure to precipitate cooperative folding of adjacent disordered domains across a length scale of ∼10 nm. Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function—for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.