Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

Background  

The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.     

渤海中、南部大型底栖动物物种多样性的研究

以1997年 6月、1998年9月和1999年4月共3个航次,覆盖渤海中、南部大部分海区的大型底栖动物丰度、生物量和生产力的研究为基础,进一步对其物种多样性进行了分析。结果表明：研究海域的大型底栖动物物种多样性没有明显的平面分布规律;物种多样性指数与沉积物因子的相关性高于其与底层水因子的相关性;沉积物中污染物含量的确会对大型底栖动物的多样性造成影响。通过对3个航次中3个相同站位的比较,发现不同年份之3个季节的平均Shannon Wiener指数无显著差异（P>0.05）;而1998年9月和1999年4月2个航次中20个相同站位的平均Shannon Wiener指数同样也未表现出明显的差异（P>0.05）。此外,渤海的物种数量要明显地高于胶州湾。     

The essential role of lipids in Alzheimer's disease

In the absence of efficient diagnostic and therapeutic tools, Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Although the precise cause of AD is still unknown, soluble β-amyloid (Aβ) oligomers are considered the proximate effectors of the synaptic injury and neuronal death occurring in the early stages of AD. Aβ oligomers may directly interact with the synaptic membrane, leading to impairment of synaptic functions and subsequent signalling pathways triggering neurodegeneration. Therefore, membrane structure and lipid status should be considered determinant factors in Aβ-oligomer-induced synaptic and cell injuries, and therefore AD progression. Numerous epidemiological studies have highlighted close relationships between AD incidence and dietary patterns. Among the nutritional factors involved, lipids significantly influence AD pathogenesis. It is likely that maintenance of adequate membrane lipid content could prevent the production of Aβ peptide as well as its deleterious effects upon its interaction with synaptic membrane, thereby protecting neurons from Aβ-induced neurodegeneration. As major constituents of neuronal lipids, n-3 polyunsaturated fatty acids are of particular interest in the prevention of AD valuable diet ingredients whose neuroprotective properties could be essential for designing preventive nutrition-based strategies. In this review, we discuss the functional relevance of neuronal membrane features with respect to susceptibility to Aβ oligomers and AD pathogenesis, as well as the prospective capacities of lipids to prevent or to delay the disease.     

The External Aldimine Form of Serine Palmitoyltransferase: STRUCTURAL, KINETIC, AND SPECTROSCOPIC ANALYSIS OF THE WILD-TYPE ENZYME AND HSAN1 MUTANT MIMICS*

Sphingolipid biosynthesis begins with the condensation of l-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a “dominant” or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg³⁷⁸ residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity.Sphingolipids are ubiquitous constituents of eukaryotic cells, where they play important roles in signaling, differentiation, and apoptosis (1–4). Defects in sphingolipid catabolism have been linked to several human diseases, such as hypertension, cancer, and disorders of the peripheral nervous system. The most common inherited peripheral neuropathy is hereditary sensory autonomic neuropathy type 1 (HSAN1).⁶ The disease leads to progressive loss of sensation in extremities and is often associated with searing pain (5–8). Genetic studies by two independent groups mapped the disease-associated mutations to the lcb1 (long chain base 1) gene on chromosome 9q22, which encodes the SPT1 subunit of serine palmitoyltransferase (SPT; EC 2.3.1.50) (9–11). SPT catalyzes the first and rate-limiting step of the sphingolipid biosynthetic pathway in all organisms studied to date (12). The reaction is a pyridoxal 5′-phosphate (PLP)-dependent, decarboxylative, Claisen condensation of the amino acid l-serine and the long chain (C16) fatty acid palmitoyl-CoA, which produces the sphingolipid precursor, 3-ketodihydrosphingosine (KDS).SPT belongs to the α-oxoamine synthase subfamily of PLP-dependent enzymes, which contains three other well characterized members: 8-amino-7-oxononanoate synthase (AONS) (13, 14), 5-aminolevulinate synthase (ALAS) (15–17), and 2-amino-3-ketobutyrate-CoA ligase (KBL) (18). These enzymes catalyze the Claisen-like condensation between an amino acid and an acyl-coenzyme A thioester (19). A common mechanism has been proposed (12, 14, 20–23) comprising the following steps: formation of an external aldimine via displacement of the lysine-PLP internal aldimine (holo-SPT) by the incoming amino acid substrate; formation of a quinonoid intermediate by abstraction of the α-proton from the PLP-amino acid external aldimine; a Claisen condensation with the fatty acid-CoA thioester substrate, followed by displacement of the CoASH to form a β-ketoacid intermediate; decarboxylation of this species to form a product quinonoid; protonation of this quinonoid to form the product external aldimine; and finally release of the α-oxoamine product and regeneration of the enzyme PLP-internal aldimine (Fig. 1). Structural studies have shown AONS, ALAS, and KBL isoforms from various species to be homodimers (13, 17, 18). In marked contrast, eukaryotic SPTs are heterodimeric, membrane-bound proteins consisting of two subunits, SPT1 and SPT2 (encoded by the lcb1 and lcb2 gene, respectively) (24).Open in a separate windowFIGURE 1.Proposed catalytic mechanism of SPT. a, the internal aldimine (holo-SPT) is displaced by l-serine to form the external adlimine; b, binding of second substrate palmitoyl-CoA causes conformational change to give the Dunathan intermediate; c, formation of the quinonoid by deprotonation of Cα hydrogen; d, thioester bond hydrolysis and release of CoASH to give β-keto acid intermediate; e, decarboxylation to form KDS product quinonoid; f, reprotonation to form KDS product external aldimine; g, KDS product release and reformation of the holo-form. The l-serine external aldimine has been trapped in the current study. The dotted line shows that steps e and f may be bypassed by an alternative mechanism that does not proceed via a product quinonoid (see Ref. 21).Analysis of a number of HSAN1 patients has revealed the four most common mutations to be C133W, C133Y, V144D, and G387A, with the cysteine mutations appearing to be the most prevalent in populations (10, 25). The impact that these mutations have on SPT activity and sphingolipid metabolism has been the focus of attention by a number of groups. Most surprisingly, these disease-associated mutations occur in the SPT1 monomer, a protein that must be inactive, since it lacks the key lysine, histidine, and aspartate residues necessary to bind and stabilize the PLP cofactor. It is the SPT2 protein that contains the conserved, active site lysine residue that forms a Schiff base internal aldimine with the PLP. However, both subunits are essential to produce functionally active SPT heterodimer (26–29), indicating that the inactive subunit has a crucial role in function. Dunn and co-workers (30) created several mutations in the yeast lcb1 and lcb2 genes, including those corresponding to the HSAN1 SPT1 mutations, and found that they dominantly inhibit SPT activity. A transgenic mouse model also revealed that the LCB1 C133W mutation led to mice with decreased tissue SPT activity and HSAN1 symptoms despite unaltered ceramide concentrations (31). A different study found that SPT activity was decreased in the tissues of HSAN1 patients, and in a Chinese hamster ovary model, hamster LCB1 C133Y and C133W mutations could not rescue cells lacking endogenous LCB1 (32). These combined studies revealed that the HSAN1 mutations act directly on the SPT enzyme but the pathological consequence on sphingolipid metabolism is unclear. Very recently, Dunn and colleagues discovered a small 80-amino acid protein (TSC3p) in yeast that stimulated yeast SPT activity, but its role is still unclear (33). Recently, a third eukaryotic subunit (SPT3) has been characterized and appears to be required for optimum SPT activity, although it is expressed most highly in placental tissue and human trophoblasts (34). It has been suggested that these three subunits could form a higher order SPT complex (35). The instability and hydrophobic nature of eukaryotic SPTs has made their isolation and therefore biophysical characterization particularly challenging (24, 36–38).We have targeted homodimeric SPT from Sphingomonas paucimobilis EY2395 to provide molecular insight into the enzymatic properties of SPT (39, 40). Recently, we reported the first high resolution x-ray crystal structure of the S. paucimobilis holo-SPT (41) and showed that the active site containing the PLP cofactor is at the dimer interface. We used this structure to model the human enzyme and map the human cysteine residue (Cys¹³³) of SPT1 onto Asn¹⁰⁰ of the bacterial SPT (41). We found that Asn¹⁰⁰ is proximal to the PLP binding site and lies at the dimer interface. To explore the effects of the HSAN1 mutations on human SPT activity and structure, we have studied mutants of the bacterial enzyme. We made the N100C mutant to better mimic native human SPT and N100W and N100Y to mimic the most common mutations in human SPT. We have characterized the mutations using kinetics, spectroscopy, and structural biology, including structures of the external l-Ser aldimine. These data provide molecular insights into the effects of HSAN1 mutations. Also, as part of these studies, we identify residues that play a role in the stabilization of intermediates in the SPT reaction and have been able to generate new insights into the substrate specificity and mechanism of the α-oxamine synthase family.     

Should We Abandon the t-Test in the Analysis of Gene Expression Microarray Data: A Comparison of Variance Modeling Strategies

High-throughput post-genomic studies are now routinely and promisingly investigated in biological and biomedical research. The main statistical approach to select genes differentially expressed between two groups is to apply a t-test, which is subject of criticism in the literature. Numerous alternatives have been developed based on different and innovative variance modeling strategies. However, a critical issue is that selecting a different test usually leads to a different gene list. In this context and given the current tendency to apply the t-test, identifying the most efficient approach in practice remains crucial. To provide elements to answer, we conduct a comparison of eight tests representative of variance modeling strategies in gene expression data: Welch''s t-test, ANOVA [1], Wilcoxon''s test, SAM [2], RVM [3], limma [4], VarMixt [5] and SMVar [6]. Our comparison process relies on four steps (gene list analysis, simulations, spike-in data and re-sampling) to formulate comprehensive and robust conclusions about test performance, in terms of statistical power, false-positive rate, execution time and ease of use. Our results raise concerns about the ability of some methods to control the expected number of false positives at a desirable level. Besides, two tests (limma and VarMixt) show significant improvement compared to the t-test, in particular to deal with small sample sizes. In addition limma presents several practical advantages, so we advocate its application to analyze gene expression data.     

Role of a conserved arginine residue during catalysis in serine palmitoyltransferase

All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.     

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.     

Diversity of the cassiicolin gene in Corynespora cassiicola and relation with the pathogenicity in Hevea brasiliensis

Corynespora cassiicola is an important plant pathogenic Ascomycete causing the damaging Corynespora Leaf Fall (CLF) disease in rubber tree (Hevea brasiliensis). A small secreted glycoprotein named cassiicolin was previously described as an important effector of C. cassiicola. In this study, the diversity of the cassiicolin-encoding gene was analysed in C. cassiicola isolates sampled from various hosts and geographical origins. A cassiicolin gene was detected in 47 % of the isolates, encoding up to six distinct protein isoforms. In three isolates, two gene variants encoding cassiicolin isoforms Cas2 and Cas6 were found in the same isolate. A phylogenetic tree based on four combined loci and elucidating the diversity of the whole collection was strongly structured by the toxin class, as defined by the cassiicolin isoform. The isolates carrying the Cas1 gene (toxin class Cas1), all grouped in the same highly supported clade, were found the most aggressive on two rubber tree cultivars. Some isolates in which no Cas gene was detected could nevertheless generate moderate symptoms, suggesting the existence of other yet uncharacterized effectors. This study provides a useful base for future studies of C. cassiicola population biology and epidemiological surveys in various host plants.