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Purpose: Cleft palate transmembrane protein 1 (Clptm1) and its paralog protein, Cisplatin resistance-related protein 9 (CRR9) constitute a highly conserved protein family, from Caenorhabditis elegans to Homo sapiens. In the present study, we examined the clinicopathological and biological significance of Clptm1 and CRR9 expression in oral squamous cell carcinoma (OSCC)., Methods: Ninety-eight OSCC tissue specimens were immunohistochemically stained with specific antibodies to Clptm1 and CRR9. The immunoreactivity of Clptm1 and CRR9 was then correlated with clinicopathological factors, including the prognosis of patients. siRNA-mediated gene silencing of CRR9 followed by cell proliferation, Matrigel invasion, anoikis assay, and gelatin zymography were performed using cultured OSCC cells. Subsequently, immunohistochemical examination including double staining was performed to determine the correlation between CRR9 and Bcl-xL expression in OSCC cells., Results: Non-tumorous oral squamous cells exhibited vague, weak, or little cytoplasmic staining with anti-Clptm1 and CRR9 antibodies. By contrast, robust Clptm1 and CRR9 immunoreactivity was found at the cancer invasion front in 55 and 54 of the 98 OSCC tissue specimens, respectively. Notably, CRR9 immunoreactivity was associated with more than 5 mm of depth of invasion, poor prognosis of the patients, and smoking habits (P < 0.05). siRNA-mediated gene silencing of CRR9 did not alter the cell proliferation but decreased Matrigel invasion and impaired anoikis resistance in cultured Ca9-22 and SAS cells. CRR9 and anti-apoptotic Bcl-xL expression levels were correlated in pT1 OSCC tissue specimens., Conclusions: Clptm1 and CRR9 were overexpressed in many OSCC tissues. In particular, CRR9 expression may promote tumor development and have a significant poor prognostic value in OSCC, possibly through conferring invasion ability and resistance to apoptotic stimuli possibly related to Bcl-xL expression. CRR9 could be a novel molecular target for patients with OSCC.

The WW domain-containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX-mediated regulation of the HIF-1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non-tumorous epithelial tissues. Moreover, TMEM207 expression was significantly correlated with lymph node metastasis and poor prognosis. An in situ proximal ligation assay demonstrated the colocalization of TMEM207 and WWOX in invasive OSCC cells, especially glycogen-rich ones. Enforced expression of TMEM207 abrogated the binding of WWOX to HIF-1α, increased HIF-1α and GLUT-1 expression, even under normoxic conditions, and promoted tumour growth in a xenoplant assay using SAS tongue squamous cancer cells. In contrast, TMEM207 knockdown decreased GLUT-1 expression in two OSCC cell lines. As a whole, our findings indicate that the aberrant expression of TMEM207 contributes to tumour progression in OSCC, possibly via promoting aerobic glycolysis., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The exfoliative toxin (ET) is a major virulence factor of Staphylococcus aureus that causes bullous impetigo and its disseminated form, staphylococcal scalded-skin syndrome (SSSS). ET selectively digests one of the intracellular adhesion molecules, desmoglein 1, of epidermal keratinocytes and causes blisters due to intraepidermal cell-cell dissociation. Most S. aureus strains that cause blistering disease produce either ETA or ETB. They are serologically distinct molecules, where ETA is encoded on a phage genome and ETB is enocded on a large plasmid. ETA-producing S. aureus strains are frequently isolated from impetigo patients, and ETB-producing S. aureus strains are isolated from SSSS. ET-induced blister formation can be reproduced with the neonatal mouse. To determine the regulatory mechanism of ET production, we investigated the role of the two-component systems and global regulators for eta or etb expression in vitro and in vivo with the mouse model. Western blot and transcription analyses using a series of mutants demonstrate ETA production was downregulated by sigB, sarS, and sarA, while ETB production was downregulated by sigB and sarA but not by sarS. Production of both toxins is upregulated by saeRS, arlRS, and agrCA. Furthermore, by the in vivo neonatal mouse model, sigB and sarS but not sarA negatively regulate the exfoliation activity of the ETA-producing strain, while sarA negatively regulates the ETB-producing strain. In both strains, saeRS, arlRS, and agrCA positively regulate the exfoliation activity in vivo. The data illustrate similar but distinct regulatory mechanisms for ETA and ETB production in S. aureus in vitro as well as in vivo.

Sister chromatid cohesion is important for the correct alignment and segregation of chromosomes during cell division. Although the cohesin complex has been shown to play a physical role in holding sister chromatids together, its loading onto chromatin is not sufficient for the establishment of sister chromatid cohesion. The activity of the cohesin complex must be turned on by Ctf7/Eco1 acetyltransferase at the replication forks as the result of a specific mechanism. To dissect this mechanism in the well established in vitro system based on the use of Xenopus egg extracts, we cloned two Xenopus orthologs of Ctf7/Eco1 acetyltransferase, XEco1 and XEco2. Both proteins share a domain structure with known members of Ctf7/Eco1 family proteins. Moreover, biochemical analysis showed that XEco2 exhibited acetyltransferase activity. We raised a specific antibody against XEco2 and used it to further characterize XEco2. In tissue culture cells, XEco2 gradually accumulated in nuclei through the S phase. In nuclei formed in egg extract, XEco2 was loaded into the chromatin at a constant level in a manner sensitive to geminin, an inhibitor of the pre-replication complex assembly, but insensitive to aphidicolin, an inhibitor of DNA polymerases. In both systems, no specific localization was observed during mitosis. In XEco2-depleted egg extracts, DNA replication occurred with normal kinetics and efficiency, and the condensation and sister chromatid cohesion of subsequently formed mitotic chromosomes was unaffected. These observations will serve as a platform for elucidating the molecular function of Ctf7/Eco1 acetyltransferase in the establishment of sister chromatid cohesion in future studies, in which XEco1 and XEco2 should be dissected in parallel.

We developed a means of quantifying proteins that have just localized in the cytoplasmic membrane using 15N-whole cell labeling together with 2D-PAGE and MALDI-TOF MS. The localization of 18 among 20 proteins consisting of 8 lipoproteins, 11 integral membrane proteins having one or two transmembrane segments and one secretory protein in the membrane fractions of Bacillus subtilis, was inhibited by the absence of SecA in a temperature-sensitive mutant. The time course of inhibition indicated that SecA participates in the localization of those proteins through immediately dependent, delayed dependent, and independent ways.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N.

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.

A novel RNA species was isolated from Bacillus subtilis, and its sequence was determined and mapped to its genetic position. This RNA was termed BS190 RNA from the length of its mature form (190 nt), and the gene encoding it is located within the aspS-yrvM intergenic region of the B. subtilis genome. Northern blotting revealed that the novel RNA species is transcribed in vegetative cells as a larger precursor (BS201 RNA, 201 nt). After transcription, the 5' end of the precursor is processed to generate the mature form, BS190 RNA. A computer-aided prediction of the secondary structure of BS190 RNA showed that it can be folded into a single hairpin structure with some bulge structures. The authors found that the growth rate of a DeltaBS190 mutant strain of B. subtilis was reduced when compared to the wild-type. A phylogenetic comparison of the sequence of the BS190 RNA gene with sequences from the databases suggests that RNA related to BS190 RNA appears to be encoded in the genomes of Bacillus halodurans and Listeria monocytogenes.

The precursors of beta-lactamase fusion proteins having the signal peptide of Bacillus subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5(*) (pPBP5(*)-BlaH6) accumulated in B. subtilis cells in the absence of SecA or Ffh. Using the five purified precursors of secretory proteins including the two fusion proteins, B. subtilis Ffh and SecA, we analyzed the protein targeting mechanism of B. subtilis in vitro. B. subtilis SecA recognized the completely translated precursors of secretory proteins to which Ffh also bound. Moreover, B. subtilis SecA-precursor complex formation was enhanced 15-to 30-fold when the precursor and Ffh were incubated first and then SecA was added, but not vice versa. We also found that B. subtilis SecA directly interacted with Ffh in vitro. These results indicate that B. subtilis SecA and Ffh interact to function cooperatively in a protein translocation pathway including other protein factors, and that Ffh, as well as SecB in Escherichia coli, enhances the binding of SecA to presecretory proteins in B. subtilis cells.

Bacillus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine-rich domain (M-domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996) J. Biol. Chem. 271, 13,140-13,146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.

We analyzed the binding activity of B. subtilis Ffh to the precursors of secretory proteins by purifying mature and precursor proteins of beta-lactamase derived from pUC18 and its derivatives, of which the signal peptide region was replaced with that of E. coli OmpA, B. subtilis AprE, PBP5* or an alkalophilic Bacillus sp. #1011 CGTase. Each of them was mixed with purified B. subtilis Ffh in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The tested precursor proteins, including those of E. coli, of which the signal sequences differ from those of B. subtilis in the number of charged amino acids and hydrophobicity, cross-linked with Ffh, whereas mature proteins did not. The addition of scRNA, the B. subtilis counterpart of mammalian SRP 7S RNA, into the mixture did not affect the complex formation. These findings suggest that B. subtilis Ffh intrinsically binds to several precursor proteins.