Exploration of the central dogma at the interface of chemistry and biology: 2010 Yale Chemical Biology Symposium

Ever since the term "central dogma" was coined in 1958, researchers have sought to control information flow from nucleic acids to proteins. Talks delivered by Drs. Anna Pyle and Hiroaki Suga at this year's Chemical Biology Symposium at Yale in May 2010 applauded recent advances in this area, at the interface between chemistry and biology.     

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to N^G-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOS^Ser1177) and Akt^Ser473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

Vernalization response of Phleum pratense and its relationships to stem lignification and floral transition

Background

Timothy is a long-day grass species well adapted for cultivation in northern latitudes. It produces elongating tillers not only in spring growth but also later in summer. As the quantity and quality of harvested biomass is dictated by canopy architecture and the proportion of stem-forming flowering tillers, the regulation of flowering is of great interest in forage grass production.

Methods

Canopy architecture, stem morphology and freezing tolerance of vernalized timothy were investigated in greenhouse and field experiments. The molecular control of development was examined by analysing the relationship between apex development and expression of timothy homologues of the floral inducer VRN1 and repressor VRN2.

Key Results

True stem formation and lignification of the sclerenchyma ring occur in both vernalized and regrowing stems irrespective of the developmental stage of the apex. The stems had, however, divergent morphology. Vernalization enhanced flowering, and the expression of the VRN1 homologue was elevated when the apex had passed into the reproductive stage. High VRN1 homologue expression was not associated with reduction in freezing tolerance and the expression coincided with increased levels of the floral repressor VRN2 homologue. Field experiments supported the observed linkage between the upregulation of the VRN1 homologue and the transition to the reproductive stage in vernalized tillers. The upregulation of putative VRN1 or VRN2 genes was restricted to vernalized tillers in the spring yield and, thus, not detected in non-vernalized tillers of the second yield; so-called regrowth.

Conclusions

The formation of a lignified sclerenchyma ring that efficiently reduces the digestibility of the stem was not related to apex development but rather to a requirement for mechanical support. The observed good freezing tolerance of reproductive timothy tillers could be one important adaptation mechanism ensuring high yields in northern conditions. Both VRN1 and VRN2 homologues required a vernalization signal for expression so the development of yield-forming tillers in regrowth was regulated independently of the studied genes.     

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 oC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.     

Unraveling the distinct distributions of VE- and N-cadherins in endothelial cells: A key role for p120-catenin

Endothelial cells express two different classical cadherins, vascular endothelial (VE) cadherin and neural (N) cadherin, having distinct functions in the vascular system. VE-cadherin is specific to endothelial adherens junctions and is strictly necessary for vascular morphogenesis. On the contrary, N-cadherin shows diffuse localization on the cell surface and interacts with mural cells for vessel stabilization. In this study, we sought to clarify the cellular mechanisms leading to the distinct cellular locations and functions of the two cadherins in the endothelium. VE-cadherin has been shown to be responsible for the junctional exclusion of N-cadherin. Using several endothelial models, we demonstrate that this property is dependent on VE-cadherin binding to p120 catenin (p120^ctn). Moreover, although in the absence of VE-cadherin N-cadherin can localize to cell contacts, angiogenesis remains impaired, demonstrating that endothelial junction formation is not sufficient for normal vessel development. Interestingly, we show that VE-cadherin, but not N-cadherin, is partially associated with cholesterol-enriched microdomains. Lipid raft-associated-VE-cadherin is characterized by a very high level of p120^ctn association, and this association is necessary for VE-cadherin recruitment into lipid rafts. Altogether, our results indicate a critical role for p120^ctn in regulating the membrane distribution of endothelial cadherins with functional consequences in terms of cadherin stabilization and intracellular signaling.     

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca²⁺ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40-200% in a concentration-dependent manner (EC₅₀ ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca²⁺ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca²⁺ sensitivity and sarcoplasmic reticulum (SR) Ca²⁺ cycling. Importantly, NCA did not affect diastolic Ca²⁺ or SR Ca²⁺ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.     

An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

The Architecture and Chemical Stability of the Archaeal Sulfolobus Turreted Icosahedral Virus

Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-Å-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-Å resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.Viruses are valuable biological tools for manipulating the cellular processes of their hosts, and they can also serve as model systems for describing macromolecular interactions through the analysis of their architecture. The Sulfolobus turreted icosahedral virus (STIV) is an archaeal virus that infects Sulfolobus solfataricus (phylum Crenarchaeota). STIV is a lytic virus that was isolated from an acidic hot spring (>80°C and pH of <3) in Yellowstone National Park (27). Hence, STIV is an important model for studying the biochemical requirements to sustain life in extreme physicochemical conditions and has the potential to become a tool for the biochemical and genetic manipulation of its host—much like bacteriophages lambda, P22, and phi29 have done for their respective hosts.Prior structural studies of STIV using cryo-electron microscopy (cryo-EM), X-ray crystallography, and proteomics have described large pentameric turret-like structures, with petal-like protrusions emanating from their central shafts (27). The T=31d capsid shell is composed of trimeric capsomers exhibiting pseudo-hexagonal symmetry, in which each of the three capsomer subunits donates two viral jelly rolls with its β-sheets normal to the capsid surface (15, 27). Capsomers surrounding the icosahedral 3-fold axes, and their neighboring subunits, make direct contact with the viral membrane via a highly basic C-terminal helix of each subunit (15, 23). Surrounding the base of the turrets are proteins that make contact with the capsid shell and a host-derived viral membrane (15). The viral membrane and the enclosed viral genome are referred to as the lipid core.The capsid architecture of STIV and the crystal structure of its major capsid protein (MCP) are strikingly similar to those of the bacteriophages PRD1, Bam35, and PM2, the alga virus PBCV-1, and the mammalian adenovirus. This similarity suggests that these viruses share an ancestral virus (2, 4, 7, 15, 25). Given the evolutionary relationship shared between STIV and PRD1, we postulated that the large turret-like vertices of STIV were used to inject the viral genome into the Sulfolobus host—a genome delivery mechanism employed by PRD1 (27).A recent report by Brumfield et al. (5) describes gross cellular ultrastructural changes induced in the Sulfolobus host during STIV infection and release. The authors identified distinct particles that appear to be assembly intermediates of STIV en route to maturation. From these intermediates the authors proposed a general mechanism of capsid assembly, in which MCP subunits and minor capsid proteins (mCPs) coassemble with the lipid membrane to form a lipid-enclosed protein vesicle. These vesicles are spherical and lack the double-stranded DNA (dsDNA) genome and turret-like appendages at the vertices.While these studies confirm an empty procapsid intermediate, the corresponding molecular mechanism associated with assembly and disassembly remains to be understood. Moreover, little is known about STIV or other archaeal virus genome delivery into the host. To obtain a better understanding of the molecular mechanism of STIV architecture and its role in genome delivery, we characterized three distinct morphologies of STIV particles using cryo-EM. An image reconstruction of one of these revealed the absence of a number of constituents decorating the STIV capsid. Hence, for simplicity, we refer to the previously reported image reconstruction (27) as “decorated” and the new image reconstruction reported here as “undecorated.” Reference-free two-dimensional (2D) class averages of the second identified morphology reveal a partially decorated STIV lipid core. The third identified morphology corresponds to the isolated STIV lipid core. Taken together, our analyses indicate that these morphologies correspond to different disassembly intermediates of STIV that can be isolated in vitro and help provide a picture of the STIV capsid architecture. Additionally, these morphologies allow us to propose an alternative possible mechanism of genome delivery.     

The potential role of mitochondrial dysfunction in seizure-associated cell death in the hippocampus and epileptogenesis

Epilepsy is considered one of the most common neurological disorders worldwide. The burst firing neurons associated with prolonged epileptic discharges could lead to a large number of changes with events of cascades at the cellular level. From its role as the cellular powerhouse, mitochondria also play a crucial role in the mechanisms of cell death. Emerging evidence has shown that prolonged seizures may result in mitochondrial dysfunction and increase of oxidative and nitrosative stress in the hippocampus that precede neuronal cell death and cause subsequent epileptogenesis. The selective dysfunction of mitochondrial respiratory chain Complex I has been suggested to be a biochemical hallmark of seizure-induced neuronal cell death and epileptogenesis. Therefore, protection of mitochondria from bioenergetic failure and oxidative stress in the hippocampus may open a new vista to the development of effective neuroprotective strategies against seizure-induced brain damage and to the design of novel treatment perspectives against therapy-resistant forms of epilepsy.