Efficient extraction of RNA from mammalian tissue

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.     

The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.     

Diversity of archaea and niche preferences among putative ammonia-oxidizing Nitrososphaeria dominating across European arable soils

Archaeal communities in arable soils are dominated by Nitrososphaeria, a class within Thaumarchaeota comprising all known ammonia-oxidizing archaea (AOA). AOA are key players in the nitrogen cycle and defining their niche specialization can help predicting effects of environmental change on these communities. However, hierarchical effects of environmental filters on AOA and the delineation of niche preferences of nitrososphaerial lineages remain poorly understood. We used phylogenetic information at fine scale and machine learning approaches to identify climatic, edaphic and geomorphological drivers of Nitrososphaeria and other archaea along a 3000 km European gradient. Only limited insights into the ecology of the low-abundant archaeal classes could be inferred, but our analyses underlined the multifactorial nature of niche differentiation within Nitrososphaeria. Mean annual temperature, C:N ratio and pH were the best predictors of their diversity, evenness and distribution. Thresholds in the predictions could be defined for C:N ratio and cation exchange capacity. Furthermore, multiple, independent and recent specializations to soil pH were detected in the Nitrososphaeria phylogeny. The coexistence of widespread ecophysiological differences between closely related soil Nitrososphaeria highlights that their ecology is best studied at fine phylogenetic scale.     

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The crystal structure of the extracellular domain of the inhibitor receptor expressed on myeloid cells IREM-1

The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands.     

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of 55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Na_v1.5; human ether-à-go-go-related gene     

Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P < 0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection.     

High yield synthesis of tentoxin, a cyclic tetrapeptide.

Tentoxin is a naturally occurring phytotoxic cyclic tetrapeptide excreted by fungi of the Alternaria alternata family. The four total syntheses of tentoxin published to date give poor total yields, mainly owing to two difficulties, the introduction of the dehydro amino acid and more especially the cyclization step. Here we describe a method that stereospecifically introduces Z-dehydrophenylalanine (deltaZPhe) by a modified Erlenmeyer aldolization reaction. The linear tetrapeptide, Boc-R1Ala-Leu-R2deltaZPhe-G1y-OMe (R1, R2: CH3, 14CH3), the precursor of tentoxin, was obtained in a 72% yield from Boc-Leu-Gly-OH. This linear tetrapeptide, labelled with carbon-14, was used for a comparative study of four cyclization reagents DPPA, DCC-PfpOH, HBTU and HATU. This last was the most effective and gave tentoxin in a 81% cyclization yield. The activated ester formed with this reagent displayed an enhanced capacity for cyclization, permitting cyclization in concentrated medium (10 mM). This new synthetic route gave tentoxin in a 60% yield from Boc-Leu-Gly-OH and offers a means of achieving the synthesis of hitherto elusive analogues.     

Analogues of tentoxin: Tools for mechanistic investigations

Summary Tentoxin [cyclo-(MeAla¹-Leu²-MeΔPhe³-Gly⁴] is a metabolite isolated from a phytopathogenic fungusAlternaria alternata, which induces chlorosis of many plants. This potential natural herbicide binds specifically to the soluble part CF₁ of the chloroplastic coupling factor, which is a proton ATP-synthase. The effect of the toxin is concentration dependent: at low concentration it is a powerful inhibitor, while at higher concentration, it stimulates the enzyme. We synthesized tentoxin and we designed new analogues in order to vary the hydrophobicity on the side chain and to study the structure activity relationships. Comparative activities suggest that it is possible to separate inhibitory and activating effects using tentoxin analogues, showing some evidence for the existence of two binding sites with different affinity constant.     

Analogues of tentoxin: Tools for mechanistic investigations

Tentoxin[cyclo-(MeAla¹-Leu²-MePhe³-Gly⁴] is a metabolite isolated from a phytopathogenic fungusAlternaria alternata, which induces chlorosis of many plants. This potentialnatural herbicide binds specifically to the soluble part CF₁of the chloroplastic coupling factor, which is a proton ATP-synthase. Theeffect of the toxin is concentration dependent: at low concentration it is apowerful inhibitor, while at higher concentration, it stimulates the enzyme.We synthesized tentoxin and we designed new analogues in order to vary thehydrophobicity on the side chain and to study the structure activityrelationships. Comparative activities suggest that it is possible toseparate inhibitory and activating effects using tentoxin analogues, showingsome evidence for the existence of two binding sites with different affinityconstant.