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PubMed2001

Direct influence of the p53 tumor suppressor on mitochondrial biogenesis and function.

Mitochondrial localization of p53 has been observed in several cell systems, but an understanding of its organelle-based physiological activity remains incomplete. The purpose of the present study was to investigate the mitochondrial DNA genomic response to dominant-negative p53 mutant miniprotein (p53DD) fused to a mitochondrial import signal. Constructs were generated to express mitochondrial targeted enhanced green fluorescent protein (mEGFP) or dominant-negative mutant p53 miniprotein (m53DD) by in-frame fusion to the signal peptide sequence of murine Cox8l. Control cytosolic vectors (cEGFP, c53DD) had the signal sequence placed in antisense orientation. NIH 3T3 cells were transiently transfected with these vectors in various combinations. Mitochondrial 16S ribosomal RNA (16S rRNA) expression and fluorochrome staining with Mitotracker Red CMXRos (DeltaPsim) were decreased in cells expressing m53DD. Both alterations were specific for mitochondrial import competence (e.g., m53DD vs. c53DD) as well as the passenger protein (e.g., m53DD vs. mEGFP). The normal functional state of mitochondria was restored with PK11195, a specific ligand of the mitochondrial peripheral-type benzodiazepine receptor. Negative dominance of m53DD on 16S rRNA expression and CMXRos staining, and rescue of these parameters with PK11195, imply a direct positive effect of p53 on mitochondrial biogenesis and function.

3T3 Cells↗

PubMed2004

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system.

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.

Cell Membrane↗

PubMed1996

Regulation of obese (ob) mRNA and plasma leptin levels in rhesus monkeys. Effects of insulin, body weight, and non-insulin-dependent diabetes mellitus.

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100-140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.

Amino Acid Sequence↗

PubMed2003

Ratjadones inhibit nuclear export by blocking CRM1/exportin 1.

In addition to previously isolated ratjadone A we describe three new members of this family, ratjadones B, C, and D, from another strain of the myxobacterium Sorangium cellulosum. We have investigated the properties of these ratjadones with respect to their activity on mammalian cell lines. We found IC(50) values in the picomolar range and a significant increase in the size of nuclei. A further examination showed that they inhibit the export of the leucine-rich nuclear export signal (LR-NES) containing proteins in different cell lines. Ratjadones are able to inhibit the formation of the nuclear export complex composed of the CRM1, RanGTP, and the cargo protein, as shown by two different in vitro assays. Finally, the binding of ratjadone C to CRM1 was demonstrated. These ratjadone activities are in the same concentration range as described for the polyketide leptomycin B (LMB) from Streptomyces sp. Like LMB, it seems that the ratjadones covalently bind to CRM1, inhibit cargo protein binding via LR-NES, and thereby block nuclear export. Thus, the ratjadones represent a new class of natural compounds which inhibit proliferation in eukaryotes by blocking nuclear export.

3T3 Cells↗

PubMed1998

Signal recognition particle-dependent targeting of ribosomes to the rough endoplasmic reticulum in the absence and presence of the nascent polypeptide-associated complex.

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

Animals↗

PubMed1998

Differences in cytotoxicity of native and engineered RIPs can be used to assess their ability to reach the cytoplasm.

Ricin is a heterodimeric cytotoxin composed of RTB, a galactose binding lectin, and RTA, an enzymatic N-glycosidase. The toxin is endocytosed, and after intracellular routing, RTA is translocated to the cytoplasm where it inactivates ribosomes resulting in a loss of host cell protein synthesis and cell death. We show for the first time that the cytotoxicity against cultured T cells by several RTA mutants is directly proportional to the enzyme activity of RTA, suggesting this is a reliable system to measure translocation effects. Large discrepancies between cytotoxicity and enzyme action for a given pair of toxins are therefore attributable to differences in cell binding, uptake, or membrane translocation. Fluid phase uptake and cytotoxicity of isolated RTA are essentially identical to that of the single chain toxin PAP. This important finding suggests that RTA, and the A chain of class 2 RIPs in general, has not evolved special translocation signals to complement the increased target cell binding facilitated by RTB. Experiments with the lectin RCA and with ebulin suggest those toxins have diminished cytotoxicity probably mediated by comparative deficiencies in B chain binding. Addition of a KDEL sequence to RTA increases fluid phase uptake, consistent with the notion that transport to the ER is important for cytotoxicity. Fusion of MBP or GST to the amino terminus of RTA has little effect on enzyme action or cytotoxicity. This result is not altered by protease inhibitors, suggesting the fusion proteins are probably not cleaved prior to translocation of the toxic A chain and implying that the toxins can carry large passenger proteins into the cytoplasm, an observation with interesting potential for analytical and therapeutic chemistry.

Amino Acid Sequence↗

PubMed1997

cDNA cloning of three cecropin-like antimicrobial peptides (Styelins) from the tunicate, Styela clava.

We cloned precursors of three new antimicrobial peptides, Styelins C, D and E, from a pharyngeal cDNA library of a tunicate, Styela clava. Preprostyelins resembled dipteran preprocecropins, while the mature domain of Styelin C resembled Cecropin P1, an antimicrobial peptide purified from the porcine intestine. Beginning with the last 6 residues of their signal sequences, Styelin C and Cecropin 1 from Drosophila virilis had 8/11 identical amino acids (72.7%). Moreover, 4 of the last 6 residues of their mature peptide domains were also identical. Styelins were shorter, by 8 residues, than dipteran cecropins and preprostyelins contained a conserved, polyanionic C-terminal extension that was absent in preprocecropins. Delineation of cecropin-like antimicrobial peptides in a protochordate supports the antiquity of this family as effectors of innate immunity in animals and it increases the likelihood that additional cecropin-like peptides will be found among other evolutionary descendants of protochordates--vertebrates.

Amino Acid Sequence↗

PubMed2003

The role of Staphylococcus aureus sortase A and sortase B in murine arthritis.

Gram-positive pathogenic bacteria display proteins on their surface that play important roles during infection. In Staphylococcus aureus, these surface proteins are anchored to the cell wall by two sortase enzymes, SrtA and SrtB, that recognize specific surface protein sorting signals. The role of sortase enzymes in bacterial virulence was examined using a murine septic arthritis model. Intravenous inoculation with any of the Delta(srtA), Delta(srtB) or Delta(srtAB) mutants resulted in significantly increased survival and significantly lower weight loss compared with the parental strain. Mice inoculated with the Delta(srtA) mutant did not express severe arthritis, while arthritis in mice inoculated with the Delta(srtB) mutant was not different from that seen in mice that were infected with the wild-type parent strain. Furthermore, persistence of staphylococci in kidneys and joints following intravenous inoculation of mice was more pronounced for wild-type and Delta(srtB) mutant strains than for Delta(srtA) or Delta(srtAB) variants. Together these results indicate that sortase B (srtB) plays a contributing role during the pathogenesis of staphylococcal infections, whereas sortase A (srtA) is an essential virulence factor for the establishment of septic arthritis.

Aminoacyltransferases↗

PubMed2003

Identification of nuclear export signals in antizyme-1.

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259-275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.

Active Transport, Cell Nucleus↗

PubMed1991

Intramolecular chaperone: the role of the pro-peptide in protein folding.

Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding.

Amino Acid Sequence↗

PubMed2002

A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1.

The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species. It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein. Here we present evidence for a new processing pathway, involving novel signal peptidase activities. The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step. In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein. The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases. Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis. A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed. Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families.

Adenosine Triphosphatases↗

PubMed2003

GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency.

Purple acid phosphatases (PAPs) are commonly found in plants, but the physiological functions of different classes of PAPs are not thoroughly understood. In the present study, we identified a novel gene, GmPAP3, from salt-stressed soybean using suppression subtractive hybridization (SSH) techniques. Protein sequence alignment studies and phylogenetic analysis strongly suggested that GmPAP3 belongs to the group of plant PAPs and PAP-like proteins that are distinct from those of fungi and animals. In addition, the invariable consensus metal binding residues of PAPs were all conserved in GmPAP3. Surprisingly, analysis of protein sorting signals showed that a putative mitochondrion targeting transit peptide is present on GmPAP3. Northern blot analysis revealed that NaCl stress causes a general induction of GmPAP3 expression in both roots and leaves of various cultivated (Glycine max) and wild (Glycine soja) soybean varieties. Further test using two genetically unrelated cultivated soybean varieties showed that the expression pattern of GmPAP3 is distinct from other PAP genes in soybeans. NaCl stress and oxidative stress but not phosphorus (P) starvation induces the expression of GmPAP3. These results suggest that the physiological role of GmPAP3 might be related to the adaptation of soybean to NaCl stress, possibly through its involvement in reactive oxygen species (ROS) forming and/or scavenging or stress-responding signal transduction pathways.

Acid Phosphatase↗

PubMed1990

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA.

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.

Adrenal Medulla↗

PubMed2006

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase.

The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.

Amino Acid Motifs↗

PubMed1999

Molecular and enzymic properties of recombinant 1, 2-alpha-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells.

For the construction of an overexpression system of the intracellular 1,2-alpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1, 2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-alpha-d-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manalpha1-3[Manalpha1-2Manalpha1-3(Manalpha1-6) Manalpha1-6]Manbeta1- 4GlcNAcbeta1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1.cm-1. Contents of the secondary structure (alpha-helix, beta-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 degrees C by differential scanning calorimetry. The calorimetric enthalpy, DeltaHcal, of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

Amino Acid Sequence↗

PubMed2006

Membrane binding of twin arginine preproteins as an early step in translocation.

The twin arginine transport (Tat) system translocates folded proteins across the bacterial inner membrane. Transport substrates are recognized by means of evolutionarily well-conserved N-terminal signal peptides. The precise role of signal peptides in the actual transport process is not yet fully understood. Potentially, much insight into the molecular details of the transport process could be gained from step-by-step in vitro experiments under controlled conditions. Here, we employ purified preproteins to study their interaction with the phospholipid membrane by using surface plasmon resonance spectroscopy. It turns out that preproteins interact tightly with a model membrane consisting of only phospholipids. This interaction, which is stabilized by both electrostatic and hydrophobic contributions, appears to constitute an early step in protein translocation by the Tat system.

Amino Acid Sequence↗

PubMed2003

Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliate Euplotes define a new family of articulins.

Protistan cells employ a wide variety of strategies to reinforce and give pattern to their outermost cortical layers. Whereas some use common cytoskeletal elements such as microtubules, others are based on novel cytoskeletal proteins that are as-yet-unknown in higher eukaryotes. The hypotrich ciliate Euplotes possesses a continuous monolayer of scales or plates, located within flattened membranous sacs ('alveoli') just below the plasma membrane, and this provides rigidity and form to the cell. Using immunological techniques, the major proteins comprising these 'alveolar plates' have been identified and termed alpha-, beta-, and gamma-plateins. The present report describes work leading to the molecular characterization of three plateins, alpha 1 and alpha 2 (predicted M(r)s of 61 and 56 kDa) and a beta/gamma form (M(r)=73 kDa). All three proteins have features that are hallmarks of articulins, a class of cytoskeletal proteins that has been identified in the cortex of a wide variety of protistan cells, including certain flagellates, ciliates, dinoflagellates and PLASMODIUM: Chief among these common features are a prominent primary domain of tandem 12-amino acid repeats, rich in valine and proline, and a secondary domain of fewer, shorter repeating units. However, variations in amino acid use within both primary and secondary repetitive domains, and a much more acidic character (predicted pIs of 4.7-4.9), indicate that the plateins represent the first proteins in a new subclass or family of articulins. This conclusion is supported by another novel feature of the plateins, the presence of a canonical hydrophobic signal peptide at the N-terminus of each derived platein sequence. This correlates well with the final cellular location of the plateins, which are assembled into plates within the membrane-limited alveolar sacs. To our knowledge, this is the first report in any eukaryote of cytoskeletal proteins with such start-transfer sequences. Confocal immunofluorescence microscopy, using antibodies to the plateins as probes, reveals that new alveolar plates (enlarging in cortical zones undergoing morphogenesis) label more faintly than mature parental plates. During plate assembly (or polymerization), the plateins thus appear to exist in a more soluble form.

Animals↗

PubMed1996

Exogenously injected nuclear import factor p10/NTF2 inhibits signal-mediated nuclear import and export of proteins in living cells.

p10/NTF2 is a cytosolic factor which is required for the translocation step in nuclear protein import in an in vitro assay with digitonin-permeabilized cells. To study the functional roles of p10/NTF2 on protein transport between the nucleus and cytoplasm in living cells, recombinant p10/NTF2 was micro-injected into cultured mammalian cells. Cytoplasmically injected p10/NTF2 strongly inhibited the nuclear import of co-injected NLS-containing substrates in a dose-dependent manner but had no effect on the diffusive import of small non-nuclear proteins. Moreover, when injected into the cell nucleus, p10/NTF2 inhibited the nuclear export of NES-containing substrates. The results suggest that the nuclear import factor p10/NTF2 may also be involved in the nuclear export of proteins and that the protein transport efficiency between the nucleus and cytoplasm may be regulated by the intracellular level of p10/NTF2.

Animals↗