Preprotein signature for full susceptibility to the co-translational translocation inhibitor cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.

The signal peptide as a new target for drug design.

Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to translocon channels across the membrane of the endoplasmic reticulum (ER) by signal peptides (SPs), which are temporary structures on the N-termini of their nascent chains. During translation, such proteins enter the lumen and membrane of the ER by a process known as co-translational translocation. Small molecules have been found that interfere with this process, decreasing protein expression by recognizing the unique structures of the SPs of particular proteins. The SP may thus become a validated target for designing drugs for numerous disorders, including certain hereditary diseases.

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents.

Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth.

The Influence of Bowel Preparation on ADC Measurements: Comparison between Conventional DWI and DWIBS Sequences.

Background and objectives: The aim of the study was to assess whether there were differences between apparent diffusion coefficient (ADC) values of diffusion-weighted imaging (DWI) and diffusion-weighted imaging with background body signal suppression (DWIBS) sequences in non-prepared and prepared bowels before and after preparation with an enteric hyperosmolar agent, to assess whether ADC measurements have the potential to avoid bowel preparation and whether ADC-DWIBS has advantages over ADC-DWI. Materials and Methods: 106 adult patients without evidence of inflammatory bowel disease (IBD) underwent magnetic resonance (MR) enterography before and after bowel preparation. ADC-DWI and ADC-DWIBS values were measured in the intestinal and colonic walls demonstrating high signal intensity (SI) at DWI tracking images of b = 800 s/mm2 before and after preparation. Results: There were significant difference (p < 0.0001) in both ADC-DWI and ADC-DWIBS results between non-prepared and prepared jejunum for DWI being 1.09 × 10-3 mm2/s and 1.76 × 10-3 mm2/s, respectively, and for DWIBS being 0.91 × 10-3 mm2/s and 1.75 × 10-3 mm2/s, respectively. Both ADC-DWI and DWIBS also showed significant difference between non-prepared and prepared colon (p < 0.0001), with DWI values 1.41 × 10-3 mm2/s and 2.13 × 10-3 mm2/s, and DWIBS-1.01 × 10-3 mm2/s and 2.04 × 10-3 mm2/s, respectively. No significant difference between ADC-DWI and ADC-DWIBS was found in prepared jejunum (p = 0.84) and prepared colon (p = 0.58), whereas a significant difference was found in non-prepared jejunum and non-prepared colon (p = 0.0001 in both samples). Conclusions: ADC between DWI and DWIBS does not differ in prepared bowel walls but demonstrates a difference in non-prepared bowel. ADC in non-prepared bowel is lower than in prepared bowel and possible overlap with the ADC range of IBD is possible in non-prepared bowel. ADC-DWIBS has no advantage over ADC-DWI in regard to IBD assessment.

Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death.

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.

Pex9p is a new yeast peroxisomal import receptor for PTS1-containing proteins.

Peroxisomal proteins carrying a type 1 peroxisomal targeting signal (PTS1) are recognized by the well-conserved cycling import receptor Pex5p. The yeast YMR018W gene encodes a Pex5p paralog and newly identified peroxin that is involved in peroxisomal import of a subset of matrix proteins. The new peroxin was designated Pex9p, and it interacts with the docking protein Pex14p and a subclass of PTS1-containing peroxisomal matrix enzymes. Unlike Pex5p, Pex9p is not expressed in glucose- or ethanol-grown cells, but it is strongly induced by oleate. Under these conditions, Pex9p acts as a cytosolic and membrane-bound peroxisome import receptor for both malate synthase isoenzymes, Mls1p and Mls2p. The inducible Pex9p-dependent import pathway provides a mechanism for the oleate-inducible peroxisomal targeting of malate synthases. The existence of two distinct PTS1 receptors, in addition to two PTS2-dependent import routes, contributes to the adaptive metabolic capacity of peroxisomes in response to environmental changes and underlines the role of peroxisomes as multi-purpose organelles. The identification of different import routes into peroxisomes contributes to the molecular understanding of how regulated protein targeting can alter the function of organelles according to cellular needs.

Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides.

The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of S. mutans Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H2O2 displayed a strong threshold behavior. Low concentrations of H2O2 had little effect on induction of comX or the bacteriocin gene cipB, but expression of these genes declined sharply if extracellular H2O2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.IMPORTANCEStreptococcus mutans inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

Antibiotic targeting of the bacterial secretory pathway.

Finding new, effective antibiotics is a challenging research area driven by novel approaches required to tackle unconventional targets. In this review we focus on the bacterial protein secretion pathway as a target for eliminating or disarming pathogens. We discuss the latest developments in targeting the Sec-pathway for novel antibiotics focusing on two key components: SecA, the ATP-driven motor protein responsible for driving preproteins across the cytoplasmic membrane and the Type I signal peptidase that is responsible for the removal of the signal peptide allowing the release of the mature protein from the membrane. We take a bird's-eye view of other potential targets in the Sec-pathway as well as other Sec-dependent or Sec-independent protein secretion pathways as targets for the development of novel antibiotics. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

A luminescent assay for real-time measurements of receptor endocytosis in living cells.

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.

Malaria impairs T cell clustering and immune priming despite normal signal 1 from dendritic cells.

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection.

Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide.

Secretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation machinery. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient versus inefficient signal sequencing, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phase event whereby the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products. Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.

A novel mouse protein differentially regulated by androgens in the submandibular and lacrimal glands.

We characterized a cDNA clone derived from the female mouse submandibular gland (SMG). The transcript of this cDNA was approximately 1.2kb in size and predicted to code a 165-amino acid protein with a putative signal peptide for a secretory pathway. This protein, named submandibular androgen-repressed protein (SMARP), had homology in the N-terminal region with members of the glutamine/glutamic acid-rich protein (GRP) family from rats. Northern blot analysis revealed that SMARP mRNA is expressed, out of the major mouse organs, only in the SMG and exorbital lacrimal gland (LG), with much more abundance in the former. For the SMG, the level of SMARP mRNA was 36 times higher in females than males, whereas for the LG it was 28 times higher in males than females. Furthermore, the level of SMARP mRNA was increased in the SMG but reduced in the LG with castration in males, whereas it was reduced in SMG but increased in LG after administration of testosterone in females or castrated males. In situ hybridization detected the signal for SMARP mRNA in the female SMG, and immunohistochemistry detected the signal for SMARP protein in the female SMG and male LG. In the female SMG, SMARP mRNA, and protein were localized intensively in a subpopulation of acinar cells, whereas in the male LG, SMARP protein was distributed diffusely in all acinar cells. These results suggested that SMARP is a secretory protein whose expression is regulated by androgens negatively in the SMG and positively in the LG.

Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth, and its activity is suppressed by MSTN propeptide (MSTNpro), the N-terminal part of MSTN precursor cleaved during post-translational MSTN processing. The current study examined which region of flatfish (Paralichthys olivaceus) MSTN-1 propeptide (MSTN1pro) is critical for MSTN inhibition. Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in Escherichia coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. A MBP-fused MSTN1pro region consisting of residues 45-100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23-265), indicating that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity. A MBP-fused MSTN1pro region consisting of residues 45-80 (Pro45-80) also showed MSTN-inhibitory activity with a lower potency, and the Pro45-80 demonstrated its MSTN binding capacity in a pull-down assay, indicating that the MSTN-inhibitory capacity of Pro45-80 is due to its binding to MSTN. Flatfish MSTN1pro synthetic peptides covering residues 45-65, 45-70, and 45-80 demonstrated MSTN-inhibitory activities, but not the synthetic peptide covering residues 45-54, indicating that residues 45-65 of flatfish MSTN1pro are essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition.

Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment.

Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D.

Streptomyces chromofuscus phospholipase D (PLD) is secreted by the bacterium and proteolytically cleaved to a more active form (PLD(37/18)) where the two parts of the molecule are still tightly associated. Based on previous sequencing results of authentic PLD(37/18), we have constructed a vector consisting of separate ORFs for the N-terminal and C-terminal portions of S. chromofuscus PLD and overexpressed active heterodimeric PLD. Neither fragment cloned separately folded properly. The identity of each peptide was confirmed by peptide-mass fingerprinting with MALDI-TOF mass spectrometry. The recombinant complex had a specific activity about six times higher than that of the recombinant intact PLD enzyme and was no longer activated by phosphatidic acid (PA). Phosphotransferase activity, binding affinity to phospholipid vesicles, loss of product activation, pH profile and pH-related Ca(2+) activation and inhibition were comparable to authentic PLD(37/18) purified from S. chromofuscus growth medium. PLD(37) alone could also be isolated; the enzyme was active but not as stable as PLD(37/18). These experimental results strongly support the hypothesis that the C-terminal peptide is necessary for correct folding and insertion of catalytic metal ions. However, they suggest the ligands involved in Fe(3+) coordination must be altered upon cleavage of the protein. Asp389, in the C-terminal fragment, whose replacement impairs Fe(3+) binding to the protein, must be replaced by another ligand, since the N-terminal fragment, once folded, is active. In the process of cloning the two peptides, the complete signal sequence for this protein was also determined. The signal peptide of S. chromofuscus PLD enzyme contained a twin arginine motif suggesting that S. chromofuscus PLD, like Bacillus subtilis phoD, is most likely secreted by the TAT translocation pathway under the transcriptional control of the pho regulon.

Mutational analyses of the signals involved in the subcellular location of DSCR1.

BACKGROUND: Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR), to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human. RESULTS: The protein's location was preferentially nuclear, independently of the isoform, cell line and insertion in the GFP's N- or C-terminal. A segment in the C-terminal, which is important in the location of the protein, was identified by deletion. On the other hand, site-directed mutational analyses have indicated the involvement of some serine and threonine residues in this event. CONCLUSION: In this paper, we discuss the identification of amino acids which can be important for subcellular location of DSCR1. The involvement of residues that are prone to phosphorylation suggests that the location and function of DSCR1 may be regulated by kinases and/or phosphatases.

Plasmodium falciparum signal sequences: simply sequences or special signals?

The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modifications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P. falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum-Golgi complex pathway.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS.

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

L-Ilf3 and L-NF90 traffic to the nucleolus granular component: alternatively-spliced exon 3 encodes a nucleolar localization motif.

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins.