Role of oxidative stress in geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex

Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor geldanamycin (GA) cytotoxicity has been attributed to the disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1 h, and cell death occurred at 6 h, inhibited by N-acetylcysteine (NAC) without preventing dissociation of proteins. At 24 h, NAC prevented cytotoxicity and Hsp90 complex disruption. However, MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring glutathione. In contrast, 24 h MEN treatment induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding protein expression, and proteasomal inhibition prevented MEN-, but not GA-induced degradation. In conclusion, whereas MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces Hsp90 complex dissociation and proteasome-independent protein degradation. These differences between MEN- and GA-induced cytotoxicity may allow more specific targeting of cancer cells.     

Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK

Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA₁ and LPA₂ that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA₁ and LPA₂ in human embryonic kidney (HEK 293) cells and that stimulation of LPA₁ receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA₁ receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA₁ and LPA₂ and have identified a novel interaction between RalA and GRK2, which is promoted by LPA₁ receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA₁ activity facilitates the formation of a novel protein complex between these two proteins.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.     

Complete genome sequence of Pirellula staleyi type strain (ATCC 27377)

Pirellula staleyi Schlesner and Hirsch 1987 is the type species of the genus Pirellula of the family Planctomycetaceae. Members of this pear- or teardrop-shaped bacterium show a clearly visible pointed attachment pole and can be distinguished from other Planctomycetes by a lack of true stalks. Strains closely related to the species have been isolated from fresh and brackish water, as well as from hypersaline lakes. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the order Planctomyces and only the second sequence from the phylum Planctobacteria/Planctomycetes. The 6,196,199 bp long genome with its 4773 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Anaerococcus prevotii type strain (PC1)

Anaerococcus prevotii (Foubert and Douglas 1948) Ezaki et al. 2001 is the type species of the genus, and is of phylogenetic interest because of its arguable assignment to the provisionally arranged family 'Peptostreptococcaceae'. A. prevotii is an obligate anaerobic coccus, usually arranged in clumps or tetrads. The strain, whose genome is described here, was originally isolated from human plasma; other strains of the species were also isolated from clinical specimen. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the genus. Next to Finegoldia magna, A. prevotii is only the second species from the family 'Peptostreptococcaceae' for which a complete genome sequence is described. The 1,998,633 bp long genome (chromosome and one plasmid) with its 1852 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Genotypes of Brassica rapa respond differently to plant-induced variation in air CO2 concentration in growth chambers with standard and enhanced venting

Growth chambers allow measurement of phenotypic differences among genotypes under controlled environment conditions. However, unintended variation in growth chamber air CO₂ concentration ([CO₂]) may affect the expression of diverse phenotypic traits, and genotypes may differ in their response to variation in [CO₂]. We monitored [CO₂] and quantified phenotypic responses of 22 Brassica rapa genotypes in growth chambers with either standard or enhanced venting. [CO₂] in chambers with standard venting dropped to 280 μmol mol^?1 during the period of maximum canopy development, ~80 μmol mol^?1 lower than in chambers with enhanced venting. The stable carbon isotope ratio of CO₂ in chamber air (δ¹³C_air) was negatively correlated with [CO₂], suggesting that photosynthesis caused observed [CO₂] decreases. Significant genotype × chamber-venting interactions were detected for 12 of 20 traits, likely due to differences in the extent to which [CO₂] changed in relation to genotypes’ phenology or differential sensitivity of genotypes to low [CO₂]. One trait, ¹³C discrimination (δ¹³C), was particularly influenced by unaccounted-for fluctuations in δ¹³C_air and [CO₂]. Observed responses to [CO₂] suggest that genetic variance components estimated in poorly vented growth chambers may be influenced by the expression of genes involved in CO₂ stress responses; genotypic values estimated in these chambers may likewise be misleading such that some mapped quantitative trait loci may regulate responses to CO₂ stress rather than a response to the environmental factor of interest. These results underscore the importance of monitoring, and where possible, controlling [CO₂].     

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX,DedD, and RlpA) in Escherichia coli Cell Constriction

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (^EFtsN) of at most 35 residues that is centered about helix H2 in the periplasm. ^EFtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (^SFtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of ^SFtsN depended on the ability of cells to constrict and, thus, on the activity of ^EFtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.Bacterial cytokinesis is mediated by a ring-shaped apparatus. Assembly of this septal ring (SR; also called the divisome or septasome) begins at the future site of fission, well before cell constriction initiates, and it remains associated with the leading edge of the invaginating cell envelope until fission is completed. The mature ring in Escherichia coli is made up of at least 10 essential division proteins (FtsA, -B, -I, -K, -L, -N, -Q, -W, and -Z and ZipA), which are each needed to prevent a lethal filamentation phenotype. The first known step in assembly of the division apparatus is polymerization of FtsZ just underneath the cytoplasmic membrane. These polymers are joined by FtsA and ZipA via direct interactions with FtsZ, resulting in an intermediate ring structure (the Z ring), onto which the remaining components assemble in a specific order to form a constriction-competent complex.In addition to the essential SR proteins, a growing number of nonessential proteins that associate with the organelle are being identified. Some of the latter are likely to serve redundant functions, while some may be required only under particular conditions (for reviews on the topic, see references 15, 19, and 25).FtsN belongs to the essential SR proteins and is thought to be the last of this class to join the organelle before the onset of cell constriction (1, 9, 11, 57, 59). It is a type II bitopic transmembrane species of 319 residues with a small cytoplasmic domain (residues 1 to 30), a single transmembrane domain (residues 31 to 54), and a large periplasmic domain (residues 55 to 319) (12) (Fig. ​(Fig.1).1). The periplasmic domain comprises three short regions with an α-helical character that are centered around residues 62 to 67 (H1), 80 to 93 (H2), and 117 to 123 (H3), an unstructured glutamine-rich linker (residues 124 to 242), and a C-terminal globular SPOR domain (residues 243 to 319) that has an affinity for peptidoglycan (55, 60) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.E. coli ftsN locus, FtsN domains, and properties of genetic constructs. Shown are the EZTnKan-2 insertion site in ftsN^slm117 strains and the deletion-replacement in ftsN<>aph (ΔftsN) strains. Numbers refer to the site of insertion (black triangle) or to the base pairs that were replaced with an aph cassette (doubleheaded arrow), counting from the start of ftsN. The domain structure of FtsN is illustrated below the ftsN gene. Indicated are the transmembrane domain (TM; light gray), helices H1, H2, and H3 (black) in the periplasmic juxtamembrane region, and the C-terminal SPOR domain (^SFtsN; dark gray). The small periplasmic peptide that is sufficient for FtsN′s essential function in cell division (^EFtsN [see text]) is indicated with the doubleheaded arrow below the domain structure diagram. Also shown are inserts present on plasmids that produce fusions of various portions of FtsN to GFP or ^TTGFP under the control of the P_lac regulatory region. ^TTGFP-fusions contain the TorA signal peptide (hatched box) that is cleaved upon export to the periplasm via the twin arginine transport (Tat) system. Columns indicate the FtsN residues present in each fusion, whether the fusion could (+) or could not (−) compensate for the absence of native FtsN, and whether it accumulated at constriction sites sharply (+++) or poorly (−−+) or appeared evenly distibuted along the periphery of the cell (−−−).As with most SR proteins, it is unclear what the essential role of FtsN is. The ftsN gene was first identified as a multicopy suppressor of a Ts allele in essential division gene ftsA (11). Elevated levels of FtsN were subsequently found to also suppress some Ts alleles in ftsI, ftsK, and ftsQ (11, 18), and even to allow the propagation of cells with a complete lack of FtsK (22, 26) or of FtsEX (48). Depletion of FtsN allows assembly of all the other known essential components into nonconstricting SRs, but the number of ring structures per unit of cell length in FtsN⁻ filaments is two- to threefold lower than in wild-type (WT) cells (9). Bacterial two-hybrid studies suggest that FtsN interacts with several other SR proteins, including FtsA, FtsI (penicillin-binding protein 3 [PBP3]), FtsQ, FtsW, and MtgA (10, 16, 17, 38). Moreover, it was recently shown that the requirement for FtsN itself can be bypassed in cells producing certain mutant forms of FtsA, which are thought to stabilize the SR to a greater degree than native FtsA (5). These observations are all compatible with a general role of FtsN in stabilizing the ring structure. In addition, it was recently found that FtsN interacts directly with PBP1B, one of the major bifunctional murein synthases in E. coli, and that it can stimulate both its transglycosylase and transpeptidase activities in vitro (46). Thus, in addition to stabilizing the SR, FtsN may have a more specific role in modulating septal murein synthesis. Lastly, based on the fact that FtsN is the last known essential protein to join the SR, it is attractive to speculate the protein plays a role in triggering the constriction phase (10, 25). To what degree any of these proposed functions contribute to the essentiality of FtsN remains unclear.What does seem clear is that the essential activity of FtsN takes place in the periplasm and that residues 139 to 319 are dispensable for its essential function (12, 55). In addition, as residues 1 to 45 are also dispensable for targeting of FtsN to division sites, some portion of the periplasmic domain must also be sufficient to direct the protein to the division apparatus (1).In a genetic screen for synthetic lethality with min (slm) (6, 7), we isolated a mutant strain carrying a transposon insertion in codon 119 of ftsN. The viability of cells containing this severely truncated ftsN^slm117 allele prompted us to better define the functional domains of FtsN, and we did so by studying the properties of fusions between various portions of FtsN to green fluorescent protein (GFP). To sublocalize a subset of these, we took advantage of the ability of the twin arginine transport system (Tat) to export functional and fluorescent GFP fusions into the periplasm, such that their periplasmic localization could be determined in live cells by fluorescence microscopy (6, 8, 50, 54).We show that the essential function of FtsN can be performed by a surprisingly small periplasmic peptide of at most 35 residues that is centered around helix H2 but that this essential domain (^EFtsN) itself is unlikely to contribute much, if anything, to the accumulation of FtsN at constriction sites. On the other hand, the nonessential periplasmic SPOR domain (^SFtsN) localized sharply to these sites by itself, while SPOR-less FtsN derivatives localized poorly, at best. Notably, septal localization of ^SFtsN depended on coproduction of ^EFtsN, in cis or in trans, unless cells were provided with the FtsA^E124A protein (5) to allow constriction to ensue in the complete absence of ^EFtsN. Localization of ^SFtsN also depended on the activity of FtsI (PBP3) and the presence of at least one of the periplasmic murein amidases, AmiA, -B, or -C. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of the protein in triggering the constriction phase of the division process. In addition, the results, taken together with earlier biochemical work (44, 46, 55), suggest that ^SFtsN is recruited to some form of septal murein that accumulates only transiently at sites of active constriction.In addition to FtsN, E. coli produces three proteins of unknown function that also bear a C-terminal SPOR domain (PF05036; Pfam 23) (20). Two of these, DamX and DedD, are inner membrane proteins with the same topology as FtsN, while the third, RlpA, is an outer membrane lipoprotein (43, 47, 53). We found that all three also accumulate at septal rings and that each of their SPOR domains act as autonomous septal targeting determinants. Moreover, phenotypes of the mutants indicate that both DamX and DedD contribute to the cell constriction process, leading to classification of these proteins as new nonessential division proteins.A SPOR domain is predicted to be present in at least 1,650 (putative) proteins from over 500 bacterial species (PF05036; Pfam 23) (20), raising the question as to how far SPOR properties have been conserved. We find that the SPOR domain of CwlC, a Bacillus subtilis murein amidase that is active during late stages of sporulation (39, 44), also accumulates sharply at division sites in E. coli.Our results predict that many other bacterial SPOR domain proteins specifically recognize the same or closely related target molecule(s) that accumulates transiently at sites of cell constriction. This is supported by a very recent study showing that SPOR domain proteins from Burkholderia thailandensis, Caulobacter crescentus, and Myxococcus xanthus accumulate at cell constriction sites as well (45).     

Glycopeptide-preferring Polypeptide GalNAc Transferase 10 (ppGalNAc T10), Involved in Mucin-type O-Glycosylation, Has a Unique GalNAc-O-Ser/Thr-binding Site in Its Catalytic Domain Not Found in ppGalNAc T1 or T2

Mucin-type O-gly co sy la tion is initiated by a large family of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer GalNAc from UDP-GalNAc to the Ser and Thr residues of polypeptide acceptors. Some members of the family prefer previously gly co sylated peptides (ppGalNAc T7 and T10), whereas others are inhibited by neighboring gly co sy la tion (ppGalNAc T1 and T2). Characterizing their peptide and glycopeptide substrate specificity is critical for understanding the biological role and significance of each isoform. Utilizing a series of random peptide and glycopeptide substrates, we have obtained the peptide and glycopeptide specificities of ppGalNAc T10 for comparison with ppGalNAc T1 and T2. For the glycopeptide substrates, ppGalNAc T10 exhibited a single large preference for Ser/Thr-O-GalNAc at the +1 (C-terminal) position relative to the Ser or Thr acceptor site. ppGalNAc T1 and T2 revealed no significant enhancements suggesting Ser/Thr-O-GalNAc was inhibitory at most positions for these isoforms. Against random peptide substrates, ppGalNAc T10 revealed no significant hydrophobic or hydrophilic residue enhancements, in contrast to what has been reported previously for ppGalNAc T1 and T2. Our results reveal that these transferases have unique peptide and glycopeptide preferences demonstrating their substrate diversity and their likely roles ranging from initiating transferases to filling-in transferases.Mucin-type O-glycosylation is a common post-translational modification of secreted and membrane-associated proteins. O-Glycan biosynthesis is initiated by the transfer of GalNAc from UDP-GalNAc to the hydroxyl groups of serine or threonine residues in a polypeptide, catalyzed by a family of polypeptide N-α-acetylgalactosaminyltransferases (ppGalNAc Ts).⁵ To date, 16 mammalian members have been reported in the literature (1–16) with a total of at least 20 members currently present in the human genome data base. Multiple members of the ppGalNAc T family have also been identified in Drosophila (9, 10, 14), Caenorhabditis elegans (3, 8), and single and multicellular organisms (17–20). Several members show close sequence orthologues across species suggesting that the ppGalNAc Ts are responsible for biologically significant functions that have been conserved during evolution. For example, in Drosophila four isoforms have close sequence orthologues to the mammalian transferases. Of the two that have been recently compared, nearly identical peptide substrate specificities have been observed between the fly and mammals, suggesting common but presently unknown functions preserved across these diverse species (21).Recently, several ppGalNAc T isoforms have been shown to be important for normal development or cellular processes. For example, inactive mutations in the fly PGANT35A (the T11 orthologue in mammals) are lethal because of the disruption of the tracheal tube structures (9, 10, 22), whereas mutations in PGANT3 alter epithelial cell adhesion in the Drosophila wing blade resulting in wing blistering (23). In humans, mutations in ppGalNAc T3 are associated with familial tumoral calcinosis, the result of the abnormal processing and secretion of the phosphaturic factor FGF23 (24, 25). Human ppGalNAc T14 has been suggested to modulate apoptotic signaling in tumor cells by its glycosylation of the proapoptotic receptors DLR4 and DLR5 (26), and very recently the specific O-glycosylation of the TGFB-II receptor (ActR-II) by the GalNTL1 has been shown to modulate its signaling in development (16).Historically, the major targets of the ppGalNAc Ts have been thought to be heavily O-glycosylated mucin domains of membrane and secreted glycoproteins. Such domains typically contain 15–30% Ser or Thr, which are highly (>50%) substituted by GalNAc. One question in the field is as follows. How is this high degree of peptide core glycosylation achieved and is it related to the large number of ppGalNAc isoforms, some of which may even have specific mucin domain preferences? Interestingly, some members of the ppGalNAc T family are known to prefer substrates that have been previously modified with O-linked GalNAc on nearby Ser/Thr residues, hence having so-called glycopeptide or filling-in activities, i.e. ppGalNAc T7 and T10 (8, 27–29). Others simply possess altered preferences against glycopeptide substrates, i.e. ppGalNAc T2 and T4 (30–33), or may be inhibited by neighboring glycosylation, i.e. ppGalNAc T1 and T2 (29, 34, 35). These latter transferases have been called early or initiating transferases, preferring nonglycosylated over-glycosylated substrates. Presently, little is known about which factors dictate the different peptide/glycopeptide specificities among the ppGalNAc Ts.The ppGalNAc Ts consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three recognizable carbohydrate-binding sites (36). Because ppGalNAc T7 and T10 prefer to transfer GalNAc to glycopeptide acceptors, it has been widely assumed that their C-terminal lectin domains would play significant roles in this activity, as has been demonstrated for other family members (27, 28, 32). Recently, Kubota et al. (37) solved the crystal structure of ppGalNAc T10 in complex with Ser-GalNAc specifically bound to its lectin domain. In this work (37), the authors further demonstrated that a T10 lectin domain mutant indeed had altered specificity against GalNAc-containing glycopeptide substrates when the acceptor Ser/Thr site was distal from the pre-existing glycopeptide GalNAc site. However, it was also observed that the lectin mutant still possessed relatively unaltered glycopeptide activity when the acceptor Ser/Thr site was directly N-terminal of a pre-existing glycopeptide GalNAc site. Kubota et al. (37) therefore concluded that for ppGalNAc T10, both its lectin and indeed its catalytic domain must contain distinct peptide GalNAc recognition sites. In support of this, Raman et al. (33) have shown that the complete removal of the ppGalNAc T10 lectin domain only slightly alters its specificity against distal glycopeptide substrates while showing no difference in its ability to glycosylate residues directly N-terminal of an existing site of glycosylation. Thus, it seems that the catalytic domain of ppGalNAc T10 may have specific requirements for a peptide O-linked GalNAc in at least the +1 position (toward the C terminus) of residues being glycosylated. As no systematic determination of the glycopeptide binding properties of the ppGalNAc Ts catalytic domain has been performed, it is unknown whether additional GalNAc peptide-binding sites exist in T10 or, for that matter, any of the other ppGalNAc Ts.We have recently reported the use of oriented random peptide substrates, GAGA(X)_nT(X)_nAGAGK (where X indicates randomized amino acid positions and n = 3 and 5) for determining the peptide substrate specificities of mammalian ppGalNAc T1, T2, and their fly orthologues (21, 38). In the present work, we extend this approach to the determination of the catalytic domain glycopeptide (Ser/Thr-O-GalNAc) substrate preferences for ppGalNAc T1, T2, and T10 employing two n = 4 oriented random glycopeptide libraries (21). Interestingly, ppGalNAc T10 displays few significant enhancements and specifically lacks the Pro residue enhancements observed for ppGalNAc T1 and T2. These findings further demonstrate the vast substrate diversity of the catalytic domains of the ppGalNAc T family of transferases.

TABLE 1

ppGalNAc transferase random substrates utilized in this workPVI, PVII, GP-I, and GP-II random (glyco)peptide substrates.