Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Functional Unfolding of ��1-Antitrypsin Probed by Hydrogen-Deuterium Exchange Coupled with Mass Spectrometry

The native state of α₁-antitrypsin (α₁AT), a member of the serine protease inhibitor (serpin) family, is considered a kinetically trapped folding intermediate that converts to a more stable form upon complex formation with a target protease. Although previous structural and mutational studies of α₁AT revealed the structural basis of the native strain and the kinetic trap, the mechanism of how the native molecule overcomes the kinetic barrier to reach the final stable conformation during complex formation remains unknown. We hypothesized that during complex formation, a substantial portion of the molecule undergoes unfolding, which we dubbed functional unfolding. Hydrogen-deuterium exchange coupled with ESI-MS was used to analyze this serpin in three forms: native, complexing, and complexed with bovine β-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of α₁AT during complex formation. A substantial portion of the α₁AT molecule unfolded transiently during complex formation, including not only the regions expected from previous structural studies, such as the reactive site loop, helix F, and the following loop, but also regions not predicted previously, such as helix A, strand 6 of β-sheet B, and the N terminus. Such unfolding of the native interactions may elevate the free energy level of the kinetically trapped native serpin sufficiently to cross the transition state during complex formation. In the current study, we provide evidence that protein unfolding has to accompany functional execution of the protein molecule.The native strain of serine protease inhibitors (serpins)¹ is considered to be crucial to their biological functions, such as plasma protease inhibition (1, 2) and hormone delivery (3). Functional execution of serpins is accompanied by the conversion of the strained native structure into a more stable conformation (4). Because some of the strained native serpin structures are spontaneously converted into a more relaxed stable latent form under physiological conditions (5–7), the native structure is not the thermodynamically most stable conformation but is a kinetically trapped conformation. Upon binding a target protease, the scissile bond of the reactive site loop (RSL) is cleaved while the protease is covalently attached to the N-terminal part of the RSL (8, 9). During the conversion of the strained structure into the stable complex conformation (Fig. 1), RSL is inserted into the central β-sheet (A sheet) between strands 3 and 5 (s3A and s5A) to form strand 4 (s4A), and the covalently attached protease is concomitantly translocated to the opposite pole (10). Serpin inhibition occurs via a suicide substrate mechanism (4, 11, 12) in which serpins, upon binding proteases, partition between cleaved serpins and stable serpin-enzyme complexes.Open in a separate windowFig. 1.Structures of native α₁AT and α₁AT-trypsin complex. Left, structure of native α₁AT (Protein Data Bank code 1QLP) illustrated with secondary structural elements (1). Right, structure of α₁AT-trypsin complex (Protein Data Bank code 1EZX). The nine α-helices are colored dark gray, and the 16 β-strands are colored light gray.As a member of the serpin family, α₁-antitrypsin (α₁AT), which serves to modulate the activity of human leukocyte elastase in the lung, has been most extensively studied with regard to both structure and inhibition mechanism. Our previous studies with stabilizing mutations of α₁AT showed that the native strain is distributed throughout the molecule and that various unfavorable structural motifs, such as hydrophobic packing, cavity in the core, and surface hydrophobic patch, appear to maintain the native strain (13, 14). Indeed stabilizing mutations localized in the region of RSL insertion during complex formation affected the inhibitory function individually by retarding the loop insertion (15). Mutations in other regions did not affect the inhibitory function individually, but collectively these mutations affected the inhibitory function when the stabilization effect reached a certain threshold (16). Maintaining the kinetic trap appears to require sustaining RSL at the hydrophobic β-barrel composed of sheet B and sheet C (B/C β-barrel) because the conversion into the stable latent conformation occurs by destabilization of the B/C β-barrel (6) as well as by the extension of RSL length (17). Thus, upon binding a target protease, RSL cleavage appears to induce a conformational conversion, and the resultant strain throughout the molecule facilitates the opening of β-sheet A and the insertion of the RSL, which is critical for the inhibitory pathway as opposed to the substrate pathway (10).Although these structural and mutational studies revealed the structural basis for maintaining the kinetic trap and its relation to the inhibitory mechanism, several questions still remain. For example, what structural changes does the native serpin molecule undergo to overcome the kinetic barrier and reach the final stable conformation during the complex formation? In the present study, to probe the structural process of overcoming the kinetic barrier during complex formation with a target protease, amide hydrogen exchange (hydrogen-deuterium exchange (H/D-EX)) was explored during the conversion of the native α₁AT to the stable complex. H/D-EX coupled with ESI-MS is a powerful analytical tool for observing protein dynamics, transient conformational changes, and protein-protein interactions (18–22). These experiments demonstrated that transient structural unfolding occurred in many regions in the α₁AT molecule during formation of the complex with β-trypsin, and some of this unfolding was unpredicted from previous structural studies.     

Mitochondrial NADH-cytochrome b(5) reductase plays a crucial role in the reduction of D-erythroascorbyl free radical in Saccharomyces cerevisiae.

The relevance of NADH-cytochrome b(5) reductase to the NADH-dependent reduction of D-erythroascorbyl free radical was investigated in Saccharomyces cerevisiae. MCR1, which is known to encode NADH-cytochrome b(5) reductase in S. cerevisiae, was disrupted by the insertion of URA3 gene into the gene of MCR1. In the mcr1 disruptant cells, the activity of NADH-D-erythroascorbyl free radical reductase almost disappeared and the intracellular level of D-erythroascorbic acid was about 11% of that of the congenic wild-type strain. In the transformant cells carrying MCR1 in multicopy plasmid, the intracellular level of D-erythroascorbic acid and the activity of NADH-D-erythroascorbyl free radical reductase increased up to 1.7-fold and 2.1-fold, respectively. Therefore, it indicated that the MCR1 product, mitochondrial NADH-cytochrome b(5) reductase, plays a key role in the NADH-dependent reduction of D-erythroascorbyl free radical in S. cerevisiae. On the other hand, the mcr1 disruptant cells were hypersensitive to hydrogen peroxide and menadione, and overexpression of MCR1 made the cells more resistant against oxidative stress. These results suggested that the mitochondrial NADH-cytochrome b(5) reductase functions as NADH-D-erythroascorbyl free radical reductase and plays an important role in the response to oxidative damage in S. cerevisiae.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Algicidal effects on Heterosigma akashiwo and Chattonella marina (Raphidophyceae), and toxic effects on natural plankton assemblages by a thiazolidinedione derivative TD49 in a microcosm

The algicidal effects of the thiazolidinedione derivative TD49 on Heterosigma akashiwo and Chattonella marina (Raphidophyceae) were assessed, and the response of the planktonic community and environment to the algicide was evaluated in a microcosm, quantifying 12 L. The abundance of over 80 % of H. akashiwo and C. marina declined in a day significantly in microcosms to which TD49 was added (final concentration 2 μM), and this was correlated with an abrupt decline in the culture pH. The number of protists (i.e., ciliates) other than H. akashiwo and C. marina gradually increased with time in the TD49 treatments, implying that the decline in numbers of H. akashiwo and C. marina cells resulting from TD49 treatment was a major factor in the growth of the other organisms. However, TD49 may be toxic to aquatic zooplankton communities, even though it is a highly selective algicide for harmful algae bloom species. The study indicates that TD49 is an effective agent for the control for H. akashiwo and C. marina blooms in enclosed and eutrophic water bodies.     

Dominance Relationship between Two Self-Incompatible<Emphasis Type="Italic">Brassica campestris</Emphasis> Haplotypes in Response to CO<Subscript>2</Subscript> Gas

InBrassica, self-incompatibility (SI) can be overcome by CO₂ application, an effective method for obtaining numerous inbred lines for F, commercial seed. We previously reported two different S-alleles ofBrassica campestris, S⁷³³ and S⁷³⁴, with extremely different degrees of susceptibility to this gas. In the current study, we raised a cross-population between those two genetic lines, and analyzed their reaction level of self-incompatibility to CO₂ (RLSICO₂). Here, all 40 of our progeny from the F₁ cross-population were susceptible, maintaining high values of RLSICO₂. This suggests that the susceptible line, S⁷³⁴, is dominant to the insusceptible line, S⁷³³. We also generated an F₂ selfing-population of each crossed progeny, S⁷³³♀ S⁷³⁴♂ and S⁷³³♂ S⁷³⁴♀, to assess the RLSICO₂ of each individual. PCR-RFLP analysis was performed to determine the S-genotype of the F₂ population. The S⁷³⁴ allele segregated in a theoretical ratio of the dominant trait, and the RLSICO₂ was consistent with the dominance relationship. Therefore, we have now demonstrated that high RLSICO₂ in β.campestris is controlled by a dominant gene. Both authors contributed equally to this work     

SMURF1 Plays a role in EGF-induced breast cancer cell migration and invasion

Epidermal growth factor (EGF) is a well-known growth factor that induces cancer cell migration and invasion. Previous studies have shown that SMAD ubiquitination regulatory factor 1 (SMURF1), an E3 ubiquitin ligase, regulates cell motility by inducing RhoA degradation. Therefore, we examined the role of SMURF1 in EGF-induced cell migration and invasion using MDA-MB-231 cells, a human breast cancer cell line. EGF increased SMURF1 expression at both the mRNA and protein levels. All ErbB family members were expressed in MDA-MB-231 cells and receptor tyrosine kinase inhibitors specific for the EGF receptor (EGFR) or ErbB2 blocked the EGF-mediated induction of SMURF1 expression. Within the signaling pathways examined, ERK1/2 and protein kinase C activity were required for EGF-induced SMURF1 expression. The overexpression of constitutively active MEK1 increased the SMURF1 to levels similar to those induced by EGF. SMURF1 induction by EGF treatment or by the overexpression of MEK1 or SMURF1 resulted in enhanced cell migration and invasion, whereas SMURF1 knockdown suppressed EGF- or MEK1-induced cell migration and invasion. EGF treatment or SMURF1 overexpression decreased the endogenous RhoA protein levels. The overexpression of constitutively active RhoA prevented EGF- or SMURF1-induced cell migration and invasion. These results suggest that EGFinduced SMURF1 plays a role in breast cancer cell migration and invasion through the downregulation of RhoA.