Novel mutations of the LolCDE complex causing outer membrane localization of lipoproteins despite their inner membrane-retention signals

In Gram-negative bacteria, lipoproteins are targeted to either the inner or outer membrane depending on their sorting signals. An ABC transporter LolCDE complex in Escherichia coli releases outer membrane-specific lipoproteins. Inner membrane-specific lipoproteins remain in the inner membrane because they each have a LolCDE-avoidance signal and therefore are not released by LolCDE. Only the LolC(A40P) mutation was previously found to cause outer membrane localization of lipoproteins despite their inner membrane-retention signals. Here, we isolated several new LolCDE mutants that cause outer membrane localization of lipoproteins possessing LolCDE-avoidance signals. Mutations were found in all three subunits of LolCDE, including the cytoplasmic ATPase subunit LolD. However, the extent of outer membrane sorting of inner membrane-specific lipoproteins differed depending on the mutation. Based on these observations, the molecular events underlying the recognition of lipoproteins by the LolCDE complex are discussed.     

The Synaptonemal Complex Shapes the Crossover Landscape Through Cooperative Assembly,Crossover Promotion and Crossover Inhibition During Caenorhabditis elegans Meiosis

The synaptonemal complex (SC) is a highly ordered proteinaceous structure that assembles at the interface between aligned homologous chromosomes during meiotic prophase. The SC has been demonstrated to function both in stabilization of homolog pairing and in promoting the formation of interhomolog crossovers (COs). How the SC provides these functions and whether it also plays a role in inhibiting CO formation has been a matter of debate. Here we provide new insight into assembly and function of the SC by investigating the consequences of reducing (but not eliminating) SYP-1, a major structural component of the SC central region, during meiosis in Caenorhabditis elegans. First, we find an increased incidence of double CO (DCO) meiotic products following partial depletion of SYP-1 by RNAi, indicating a role for SYP-1 in mechanisms that normally limit crossovers to one per homolog pair per meiosis. Second, syp-1 RNAi worms exhibit both a strong preference for COs to occur on the left half of the X chromosome and a significant bias for SYP-1 protein to be associated with the left half of the chromosome, implying that the SC functions locally in promoting COs. Distribution of SYP-1 on chromosomes in syp-1 RNAi germ cells provides strong corroboration for cooperative assembly of the SC central region and indicates that SYP-1 preferentially associates with X chromosomes when it is present in limiting quantities. Further, the observed biases in the distribution of both COs and SYP-1 protein support models in which synapsis initiates predominantly in the vicinity of pairing centers (PCs). However, discontinuities in SC structure and clear gaps between localized foci of PC-binding protein HIM-8 and X chromosome-associated SYP-1 stretches allow refinement of models for the role of PCs in promoting synapsis. Our data suggest that the CO landscape is shaped by a combination of three attributes of the SC central region: a CO-promoting activity that functions locally at CO sites, a cooperative assembly process that enables CO formation in regions distant from prominent sites of synapsis initiation, and CO-inhibitory role(s) that limit CO number.REDUCTION in ploidy during sexual reproduction depends on the ability to form pairwise associations between homologous chromosomes. The homolog pairing process typically culminates in an arrangement in which the homologs are aligned in parallel along their lengths, with a highly ordered proteinaceous structure known as the synaptonemal complex (SC) located at the interface between them. Further, in most organisms, pairwise associations between homologs are solidified through the formation of crossovers (COs) between their DNA molecules, a process that is completed within the context of the SC.The SC has long been recognized as a hallmark cytological feature of meiosis. It was discovered on the basis of its highly ordered structure and location at the interface between aligned chromosomes in electron microscopy images of nuclei at the pachytene stage of meiotic prophase (Moses 1956, 2006). Each of the homologs is associated with one of the two lateral elements (LEs) of the SC, which are composed of cohesin complexes and other meiosis-specific structural and regulatory proteins (reviewed in Mlynarczyk-Evans and Villeneuve 2010). The LEs are connected by a highly ordered latticework of transverse filaments, and often a pronounced central element, that comprise the central region of the SC. The protein components of the SC central region are very poorly conserved at the primary sequence level, but the major central region proteins identified from diverse species share in common extended regions of predicted coiled coil structure.The SC has been demonstrated to have at least two conserved functions in meiotic prophase. First, the SC serves to stabilize and maintain tight associations along the lengths of aligned homologs (reviewed in Mlynarczyk-Evans and Villeneuve 2010). This is true both in organisms in which SC assembly is coupled to formation of recombination intermediates (e.g., budding yeast, mouse, and Arabidopsis) and in organisms in which formation of SC between homologs can occur independently of recombination (e.g., Caenorhabditis elegans and Drosophila). Second, SC central region proteins play a role in promoting maturation of recombination intermediates into crossover products (reviewed in De Boer and Heyting 2006). How the SC functions to promote CO formation is not well understood. Moreover, whether the SC might also have additional functions that help to ensure a successful outcome of meiosis has been a matter of debate.In addition to its roles in stabilization of pairing and promoting CO formation, the SC has also been proposed to function in inhibiting CO formation (Egel 1978, 1995; Maguire 1988). This idea of the SC playing an inhibitory role in recombination dates almost as far back as the discovery of the SC itself. Finding a highly ordered structure with a zipper-like appearance extending along the length of each homolog pair naturally gave rise to speculation that it might play a role in the phenomenon of crossover interference, defined as the ability of a (nascent) CO to inhibit the formation of other COs nearby on the same chromosome pair (Muller 1916; Hillers 2004). It was variously proposed either that the SC might serve as a conduit of information along a chromosome pair (e.g., undergoing a distance-dependent “change in state” to inhibit COs) or that SC polymerization might itself confer CO inhibition (Egel 1978; Maguire 1988; Sym and Roeder 1994).Early analysis of the budding yeast mutants lacking Zip1, a major structural component of the SC central region, initially seemed to support the idea that the SC central region played a key role in CO interference, as zip1 mutants formed COs at 30–50% of wild-type levels and the residual COs did not display interference (Sym and Roeder 1994). However, these data were subsequently reinterpreted by postulating that the major interference-sensitive meiotic CO pathway is eliminated in the zip1 mutant and that the residual COs form by an alternative pathway that is not subject to interference (Zalevsky et al. 1999; de los Santos et al. 2003). According to this two-pathway view, the lack of interference in the zip1 mutant can be readily explained without invoking a role for Zip1 in the interference mechanism per se. Conversely, Page and Hawley found that Drosophila females expressing a mutant form of the fly SC central region protein C(3)G retained substantial interference between residual COs despite exhibiting incomplete synapsis, implying that complete SC formation was not required for CO interference (Page and Hawley 2001). In light of these and other findings (e.g., Borner et al. 2004; Fung et al. 2004), the idea that the SC might play a role in inhibiting CO formation fell from favor.In this study, we revisit a potential role for the SC central region in inhibiting CO formation, using the C. elegans experimental system. Several features make this an interesting system for investigating factors that promote and/or inhibit COs during meiosis. First, essentially all COs in C. elegans depend on conserved meiotic CO-promoting machinery (i.e., Msh4 and Msh5) and on SC central region proteins (SYP-1, -2, -3, and -4), so analysis is generally not complicated by residual COs forming by alternative pathways (Zalevsky et al. 1999; Kelly et al. 2000; MacQueen et al. 2002; Colaiacovo et al. 2003; Smolikov et al. 2007a, 2009). Second, C. elegans hermaphrodites exhibit robust CO control, with COs usually being limited to one per homolog pair per meiosis (Hillers and Villeneuve 2003; Nabeshima et al. 2004; Hammarlund et al. 2005). Consequently, circumstances that give rise to double crossover (DCO) meiotic products can be inferred to represent impairment of mechanisms that normally inhibit CO formation. Finally, COs are distributed nonuniformly along the lengths of the chromosomes, with each chromosome containing broad domains of relatively high CO frequency flanking a more central domain where CO frequency is low (Brenner 1974; Barnes et al. 1995; Rockman and Kruglyak 2009), providing an opportunity to evaluate how factors that promote and/or inhibit COs contribute to this landscape.Our strategy was to use RNAi to reduce the levels of wild-type SYP-1 protein without eliminating synapsis entirely and then to examine the effects on CO frequency and distribution. This approach indeed revealed a role for SC central region protein SYP-1 in mechanisms that normally limit the number of COs per homolog pair. Further, it also revealed a role for the SC central region in determining CO distribution, presumably by enabling formation of COs in chromosome regions distant from the dominant site of synapsis initiation. Finally, our experimental design also afforded us the opportunity to evaluate spatial distribution of the SC in the context of limiting amounts of a key central region component. This analysis provided additional insight into the process of SC assembly and the role of cis-acting meiotic pairing centers in this process.     

Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors

A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure–activity relationship (SAR) obtained in this work.     

Genetic encoding of non‐natural amino acids in Drosophila melanogaster Schneider 2 cells

Insect cells are useful for the high‐yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell‐based system for incorporating non‐natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA^Tyr in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl‐tRNA synthetase specific for 3‐iodo‐L ‐tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non‐natural amino acids, and the “transient” method generated a sevenfold higher yield. By this method, 4‐azido‐L ‐phenylalanine was incorporated into human interleukin‐8 at a specific site. The yield of the azido‐containing IL‐8 was 1 μg/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger–Bertozzi reaction.     

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17°C and 23°C. However, sex ratios become female-biased at <15°C and male-biased at 25°C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.     

Leptodora kindtii: a cladoceran species highly sensitive to toxic chemicals

Leptodora kindtii is a major predator of small zooplankton in eutrophic and fish-abundant lakes. However, as it is very difficult to culture in the laboratory, information about its sensitivity to pollutants is lacking. We have successfully established a laboratory clonal culture of Leptodora. In this study, acute toxicities of an insecticide and three heavy metals to Leptodora were estimated by using laboratory-cultured individuals. Our results suggest that Leptodora is more susceptible to contamination with those chemicals than the standard test organism, Daphnia.     

Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.     

Nucleoredoxin Negatively Regulates Toll-like Receptor 4 Signaling via Recruitment of Flightless-I to Myeloid Differentiation Primary Response Gene (88)

We previously characterized nucleoredoxin (NRX) as a negative regulator of the Wnt signaling pathway through Dishevelled (Dvl). We perform a comprehensive search for other NRX-interacting proteins and identify Flightless-I (Fli-I) as a novel NRX-binding partner. Fli-I binds to NRX and other related proteins, such as Rod-derived cone viability factor (RdCVF), whereas Dvl binds only to NRX. Endogenous NRX and Fli-I in vivo interactions are confirmed. Both NRX and RdCVF link Fli-I with myeloid differentiation primary response gene (88) (MyD88), an important adaptor protein for innate immune response. NRX and RdCVF also potentiate the negative effect of Fli-I upon lipopolysaccharide-induced activation of NF-κB through the Toll-like receptor 4/MyD88 pathway. Embryonic fibroblasts derived from NRX gene-targeted mice show aberrant NF-κB activation upon lipopolysaccharide stimulation. These results suggest that the NRX subfamily of proteins forms a link between MyD88 and Fli-I to mediate negative regulation of the Toll-like receptor 4/MyD88 pathway.