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About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo , revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells., (© 2018 Fujimoto et al.)

Somites are a pair of epithelial spheres beside a neural tube and are formed with an accurate periodicity during embryogenesis in vertebrates. It has been known that Hes7 is one of the core clock genes for somitogenesis, and its expression domain is restricted in the presomitic mesoderm (PSM). However, the molecular mechanism of how Hes7 transcription is regulated is not clear. Here, using transgenic mice and luciferase-based reporter assays and in vitro binding assays, we unravel the mechanism by which Hes7 is expressed exclusively in the PSM. We identified a Hes7 essential region residing -1.5 to -1.1 kb from the transcription start site of mouse Hes7 , and this region was indispensable for PSM-specific Hes7 expression. We also present detailed analyses of cis -regulatory elements within the Hes7 essential region that directs Hes7 expression in the PSM. Hes7 expression in the PSM was up-regulated through the E-box, T-box, and RBPj-binding element in the Hes7 essential region, presumably through synergistic signaling involving mesogenin1, T-box6 (Tbx6), and Notch. Furthermore, we demonstrate that Tbx18, Ripply2, and Hes7 repress the activation of the Hes7 essential region by the aforementioned transcription factors. Our findings reveal that a unified transcriptional regulatory network involving a Hes7 essential region confers robust PSM-specific Hes7 gene expression., (© 2018 Hayashi et al.)

Microsomal triglyceride transfer protein (MTP) is a target to reduce plasma lipids because of its indispensable role in triglyceride-rich lipoprotein biosynthesis. MTP inhibition in Western diet fed mice decreased plasma triglycerides/cholesterol, whereas increasing plasma alanine/aspartate aminotransferases (ALT/AST) and hepatic triglycerides/free cholesterol. Free cholesterol accumulated in the endoplasmic reticulum (ER) and mitochondria resulting in ER and oxidative stresses. Mechanistic studies revealed that MTP inhibition increased transcription of the GPT/GOT1 genes through up-regulation of the IRE1α/cJun pathway leading to increased synthesis and release of ALT1/AST1. Thus, transcriptional up-regulation of GPT/GOT1 genes is a major mechanism, in response to ER stress, elevating plasma transaminases. Increases in plasma and tissue transaminases might represent a normal response to stress for survival.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

Linker histone H1 has been generally viewed as a global repressor of transcription by preventing the access of transcription factors to sites in chromatin. However, recent studies suggest that H1 can interact with other regulatory factors for its action as a negative modulator of specific genes. To investigate these aspects, we established a human cell line expressing H1.2, one of the H1 subtypes, for the purification of H1-interacting proteins. Our results showed that H1.2 can stably associate with sets of cofactors and ribosomal proteins that can significantly repress p53-dependent, p300-mediated chromatin transcription. This repressive action of H1.2 complex involves direct interaction of H1.2 with p53, which in turn blocks p300-mediated acetylation of chromatin. YB1 and PURalpha, two factors present in the H1.2 complex, together with H1.2 can closely recapitulate the repressive action of the entire H1.2 complex in transcription. Chromatin immunoprecipitation and RNA interference analyses further confirmed that the recruitment of YB1, PURalpha, and H1.2 to the p53 target gene Bax is required for repression of p53-induced transcription. Therefore, these results reveal a previously unrecognized function of H1 as a transcriptional repressor as well as the underlying mechanism involving specific sets of factors in this repression process.

The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.

The recently characterized enzyme NEIL2 (Nei-like-2), one of the four oxidized base-specific DNA glycosylases (OGG1, NTH1, NEIL1, and NEIL2) in mammalian cells, has poor base excision activity from duplex DNA. To test the possibility that one or more proteins modulate its activity in vivo, we performed mass spectrometric analysis of the NEIL2 immunocomplex and identified Y box-binding (YB-1) protein as a stably interacting partner of NEIL2. We show here that YB-1 not only interacts physically with NEIL2, but it also cooperates functionally by stimulating its base excision activity by 7-fold. Moreover, YB-1 interacts with the other NEIL2-associated BER proteins, namely, DNA ligase III alpha and DNA polymerase beta and thus could form a large multiprotein complex. YB-1, normally present in the cytoplasm, translocates to the nucleus during UVA-induced oxidative stress, concomitant with its increased association with and activation of NEIL2. NEIL2-initiated base excision activity is significantly reduced in YB-1-depleted cells. YB-1 thus appears to have a novel regulatory role in NEIL2-mediated repair under oxidative stress.

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. beta-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1(-/-) mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1(-/-) embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1(-/-) mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.

P-glycoprotein, encoded by the multidrug resistance (MDR)-1 gene, expels various drugs from cells resulting in multidrug resistance. We found previously that interleukin-2, a lymphocyte-activation cytokine, induces P-glycoprotein expression on lymphocytes. Lymphocyte activation involves adhesion with the extracellular matrix, such as hyaluronan, through adhesion molecules on lymphocytes. We investigated the transcriptional regulation of MDR-1 in lymphocytes by fragmented hyaluronan. Fragmented hyaluronan (especially the 6.9-kDa form), not native high molecular hyaluronan, induced translocation of YB-1, a specific transcriptional factor for MDR-1, from the cytoplasm into the nucleus and resulted in the transcription of MDR-1 and the expression of P-glycoprotein on lymphocytes in a dose-dependent manner. Transfection of YB-1 antisense oligonucleotides inhibited P-glycoprotein expression induced by fragmented hyaluronan. The fragmented hyaluronan induced significant P-glycoprotein expression on only activated CD4+ T cells, which highly expressed CD69, and resulted in excretion of intracellular dexamethasone added in vitro. Cyclosporin A, a competitive P-glycoprotein inhibitor, restored intracellular dexamethasone levels in CD4+ T cells. Anti-CD44 monoclonal antibody (Hermes-1) inhibited fragmented hyaluronan-induced YB-1 activation and P-glycoprotein expression in CD4+ T cells. We provide the first evidence that binding of fragmented hyaluronan to CD44 induces YB-1 activation followed by P-glycoprotein expression in accordance with activation of CD4+ T cells. Our findings imply that CD4+ T cell activation by fragmented hyaluronan, induced by characteristic extracellular matrix changes in inflammation, tumors, and other conditions, results in P-glycoprotein-mediated multidrug resistance.

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.

Drug-induced modifications of transcription factors play important roles in both apoptosis and survival signaling. The data presented here show that the DNA topoisomerase II poison TAS-103 transactivated the SV40 promoter in a GC-box-dependent manner and induced Sp1 acetylation in cells expressing p300. This activity was not observed in cells lacking p300. TAS-103 treatment also enhanced the p300 content of the nucleus and the interaction of p300 with Sp1. Cellular susceptibility to TAS-103 was correlated with p300 expression but not with topoisomerase II expression. Furthermore, the presence of p300 significantly sensitized cancer cells to TAS-103 but not to cisplatin. Taken together, these findings demonstrate novel genomic responses to anticancer agents that modulate Sp1 acetylation and Sp1-dependent transcription in an apoptotic pathway.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a severe IgE-mediated allergic reaction provoked by the combination of wheat-ingestion with intensive physical exercise over the next few hours. Among wheat proteins, omega-5 gliadin, which is one of the components of fast omega-gliadin, has been reported as a major allergen in the anaphylaxis. In this study, we detected IgE-binding epitopes within the primary sequence of omega-5 gliadin using arrays of overlapping peptides synthesized on derivatized cellulose membranes. Sera from four patients with WDEIA having specific IgE to the fast omega-gliadin were used to probe the membrane. Seven epitopes, QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, and PYPP, were detected within the primary sequence of omega-5 gliadin. By using sera of 15 patients, 4 of them, QQIPQQQ, QQFPQQQ, QQSPEQQ, and QQSPQQQ, were found to be dominant epitopes. Mutational analysis of the QQIPQQQ and QQFPQQQ indicated that amino acids at positions Gln(1), Pro(4), Gln(5), Gln(6), and Gln(7) were critical for IgE binding. These results will provide a useful tool for developing safer wheat products in addition to diagnostic and immunotherapy techniques for WDEIA.

In Saccharomyces cerevisiae, two highly conserved proteins, Rvb1p/Tih1p and Rvb2p/Tih2p, have been demonstrated to be major components of the chromatin-remodeling INO80 complex. The mammalian orthologues of these two proteins have been shown to physically associate with the TATA-binding protein (TBP) in vitro but not clearly in vivo. Here we show that yeast proteins interact with TBP under both conditions. To assess the functional importance of these interactions, we examined the effect of mutating both TIH2/RVB2 and SPT15, which encodes TBP, on yeast cell growth. Intriguingly, only those spt15 mutations that affected the ability of TBP to bind to the TATA box caused synthetic growth defects in a tih2-ts160 background. This suggests that Tih2p might be important in recruiting TBP to the promoter. A DNA microarray technique was used to identify genes differentially expressed in the tih2-ts160 strain grown at the restrictive temperature. Only 34 genes were significantly and reproducibly affected; some up-regulated and others down-regulated. We compared the transcription of several of these Tih2p target genes in both wild type and various mutant backgrounds. We found that the transcription of some genes depends on functions possessed by both Tih2p and TBP and that these functions are substantially impaired in the spt15/tih2-ts160 double mutants that confer synthetic growth defects.

Several endoplasmic reticulum (ER)-resident luminal proteins have a characteristic ER retrieval signal, KDEL, or its variants at their C terminus. Our previous work searching EST databases for proteins containing the C-terminal KDEL motif predicted some novel murine proteins, one of which designated JPDI (J-domain-containing protein disulfide isomerase-like protein) is characterized in this study. The primary structure of JPDI is unique, because in addition to a J-domain motif adjacent to the N-terminal translocation signal sequence, four thioredoxin-like motifs were found in a single polypeptide. As examined by Northern blotting, the expression of JPDI was essentially ubiquitous in tissues and almost independent of ER stress. A computational prediction that JPDI is an ER-resident luminal protein was experimentally supported by immunofluorescent staining of epitope-tagged JPDI-expressing cells together with glycosylation and protease protection studies of this protein. JPDI probably acts as a DnaJ-like partner of BiP, because a recombinant protein carrying the J-domain of JPDI associated with BiP in an ATP-dependent manner and enhanced its ATPase activity. We speculate that for the folding of some proteins in the ER, chaperoning by BiP and formation of proper disulfide bonds may synchronously occur in a JPDI-dependent manner.

We recently reported that c-Myc represses the transcription of platelet-derived growth factor (PDGF) beta-receptor (Izumi, H., Molander, C., Penn, L. Z., Ishisaki, A., Kohno, K., and Funa, K. (2001) J. Cell Sci. 114, 1533-1544). We demonstrate here that the p53 family protein p73alpha represses PDGF beta-receptor transcription essentially by the same mechanism. p73alpha but not p73beta or p53 represses the transcription in concordance with its ability to bind NF-YC and NF-YB. None of other p73 isoforms (i.e. p73beta, p73gamma, p73epsilon), C-terminal deletion mutants of p73alpha, and p53 is able to bind NF-Y with the exception of p63alpha. This finding suggests that the sterile alpha-motif domain present only in p73alpha and p63alpha is the interaction site. For the repression, the N-terminal transactivation domain of p73alpha is also indispensable, arguing for the importance of the activity of p73alpha in the mechanism. p73alpha binds the C-terminal HAP domain of NF-YC previously found to be the interaction site with c-Myc and TBP. Because c-Myc induces and activates p73alpha (Zaika, A., Irwin, M., Sansome, C., and Moll, U. M. (2001) J. Biol. Chem. 276, 11310-11316) and they bind each other (Uramoto, H., Izumi, H., Ise, T., Tada, M., Uchiumi, T., Kuwano, M., Yasumoto, K., Funa, K., and Kohno, K. (2002) J. Biol. Chem. 277, in press), we examined whether the repression by p73 is dependent on c-Myc. However, Myc-null rat fibroblasts are also susceptible to p73alpha-induced repression. Serum stimulation of NIH3T3 cells gradually decreased the amount of endogenous NF-Y binding to the PDGF beta-receptor promoter, whereas NF-YA expression in the nuclear extracts remains unchanged. Our results indicate that serum stimulation induces c-Myc and p73alpha, leading to the down-regulation of PDGF beta-receptor expression by repressing its transcription.

We have isolated two overlapping genomic clones that contain the 5'-terminal portion of the human vacuolar H(+)-ATPase c subunit (ATP6L) gene. The sequence preceding the transcription initiation site, which is GC-rich, contains four GC boxes and one Oct1-binding site, but there is no TATA box or CCAAT box. In vivo footprint analysis in human cancer cells shows that two GC boxes and the Oct1-binding site are occupied by Sp1 and Oct1, respectively. We show here that treatment with anticancer agents enhances ATP6L expression. Although cisplatin did not induce ATP6L promoter activity, it altered ATP6L mRNA stability. On the other hand, the DNA topoisomerase II inhibitor, TAS-103, strongly induced promoter activity, and this effect was completely eradicated when a mutation was introduced into the Oct1-binding site. Treatment with TAS-103 increased the levels of both Sp1/Sp3 and Oct1 in nuclear extracts. Cooperative binding of Sp1 and Oct1 to the promoter is required for promoter activation by TAS-103. Incubation of a labeled oligonucleotide probe encompassing the -73/-68 GC box and -64/-57 Oct1-binding site with a nuclear extract from drug-treated KB cells yielded higher levels of the specific DNA-protein complex than an extract of untreated cells. Thus, the two transcription factors, Sp1 and Oct1 interact, in an adaptive response to DNA damage, by up-regulating expression of the vacuolar H(+)-ATPase genes. Furthermore, combination of the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A1, with TAS-103 enhanced apoptosis of KB cells with an associated increase in caspase-3 activity. Our data suggest that the induction of V-ATPase expression is an anti-apoptotic defense, and V-ATPase inhibitors in combination with low-dose anticancer agents may provide a new therapeutic approach.

YB-1 is a member of the cold shock domain family of proteins that is important for signaling DNA damage and cell proliferation. YB-1 is induced by DNA damage and can also recognize cisplatin-modified DNA. In this study we observed a 6-fold increase in the steady-state level of YB-1 mRNA in response to cisplatin exposure in cells of the human cancer cell line KB. We present evidence from cotransfection experiments for a critical role of c-Myc and p73 in the transactivation of the YB-1 promoter. p73 transactivated the YB-1 promoter in experiments with Saos-2 cells, which express c-Myc, but not with HO15.19 cells, which lack c-Myc. In turn, c-Myc transactivated an intact YB-1 promoter but not a YB-1 promoter with a mutant E-box, indicating that the E-box is necessary for the response of the promoter to cisplatin. We also found that p73 interacts with c-Myc in vitro and in vivo. Using deletion mutants we showed that the DNA-binding domain of p73 and the C-terminal region of c-Myc are required for the interaction. Furthermore, p73 stimulated the interaction of Max with c-Myc and promoted binding of the c-Myc-Max complex to its target DNA. Our data suggest that p73 stimulates the transcription of the YB-1 promoter by enhancing recruitment of the c-Myc-Max complex to the E-box.

Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics. They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins. The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized. Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass. Nse1p is essential for cell proliferation and localizes primarily in the nucleus. nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype. These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair. We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation.

The enterohepatic circulation is essential for the maintenance of bile acids and cholesterol homeostasis. The ileal bile acid transporter on the apical membrane of enterocytes mediates the intestinal uptake of bile salts, but little is known about the bile salt secretion from the basolateral membrane of enterocytes into blood. In the basolateral membrane of enterocytes, an ATP-binding cassette transporter, multidrug resistance protein 3 (MRP3), is expressed, which has the ability to transport bile salts. We hypothesized that MRP3 might play a role in the enterohepatic circulation of bile salts by transporting them from enterocytes into circulating blood through the up-regulation of MRP3 expression, so we investigated the transcriptional control of MRP3 in response to bile salts. MRP3 mRNA levels were increased about 3-fold in human colon cells by chenodeoxycholic acid (CDCA), in a dose- and time-dependent manner. In the promoter assay, the promoter activity of MRP3 was increased about 3-fold over the basal promoter activity when treated with CDCA, and the putative bile salt-responsive elements exist in the region -229/-138 including two alpha-1 fetoprotein transcription factor (FTF)-like elements. Constructs with a specific mutation in the consensus sequence of FTF elements showed no increase in basal transcriptional activity following CDCA treatment. In electrophoretic mobility shift assay with nuclear extracts, specific binding of FTF to FTF-like elements was observed when treated with CDCA. The expression of FTF mRNA levels were also markedly enhanced in response to CDCA, and overexpression of FTF specifically activated the MRP3 promoter activity about 4-fold over the basal promoter activity. FTF thus might play a key role not only in the bile salt synthetic pathway in hepatocytes but also in the bile salt excretion pathway in enterocytes through the regulation of MRP3 expression. MRP3 may contribute as a plausible bile salt-exporting transporter to the enterohepatic circulation of bile salts.

A nonhistone chromosomal protein, high mobility group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds preferentially to cisplatin-modified DNA. HMG1 also functions as a coactivator of p53, a tumor suppressor protein. We investigated physical interactions between HMG1 and p53 and the influence of p53 on the ability of HMG1 to recognize damaged DNA. Using immunochemical coprecipitation, we observed binding of HMG1 and p53. Interaction between HMG1 and p53 required the HMG A box of HMG1 and amino acids 363-376 of p53. Cisplatin-modified DNA binding by HMG1 was significantly enhanced by p53. An HMG1-specific antibody that recognized the A box of this protein also stimulated cisplatin-modified DNA binding. These data suggest that an interaction with either p53 or antibody may induce conformational change in the HMG1 A box that optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and p53-mediated DNA repair.

Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant, tih2-160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G(1) phase. Northern blot analysis revealed that Tih2p is required for transcription of G(1) cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2-160 and those of taf145 mutants suggest a role for TIH2 in the regulation of RNA polymerase II-directed transcription.

The COPII coat is required for vesicle budding from the endoplasmic reticulum (ER), and consists of two heterodimeric subcomplexes, Sec23p/Sec24p, Sec13p/Sec31p, and a small GTPase, Sar1p. We characterized a yeast mutant, anu1 (abnormal nuclear morphology) exhibiting proliferated ER as well as abnormal nuclear morphology at the restrictive temperature. Based on the finding that ANU1 is identical to SEC24, we confirmed a temperature-sensitive protein transport from the ER to the Golgi in anu1-1/sec24-20 cells. Overexpression of SFB2, a SEC24 homologue with 56% identity, partially suppressed not only the mutant phenotype of sec24-20 cells but also rescued the SEC24-disrupted cells. Moreover, the yeast two-hybrid assay revealed that Sfb2p, similarly to Sec24p, interacted with Sec23p. In SEC24-disrupted cells rescued by overexpression of SFB2, some cargo proteins were still retained in the ER, while most of the protein transport was restored. Together, these findings strongly suggest that Sfb2p functions as the component of COPII coats in place of Sec24p, and raise the possibility that each member of the SEC24 family of proteins participates directly and/or indirectly in cargo-recognition events with its own cargo specificity at forming ER-derived vesicles.

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) enhances expression of interleukin-6, collagenase, plasminogen activator inhibitor-1, and basic fibroblast growth factor genes in human omental microvascular endothelial (HOME) cells in culture. In this study, we found that treatment of HOME cells with TNF-alpha or interleukin-1 (IL-1) caused enhanced expression of low density lipoprotein (LDL) receptor. A few-fold increase in both LDL binding activity and the receptor mRNA levels was observed when HOME cells were treated with either TNF-alpha or IL-1. Northern blot analysis showed that cellular expression of LDL receptor gene was significantly increased 12-24 h after exposure to TNF-alpha. No significant changes in the life-span of LDL receptor mRNA were observed in untreated and TNF-alpha-treated cells. Scatchard analysis showed an increased receptor number for LDL in TNF-alpha-treated cells. Parallel to increased LDL binding activity, internalization and degradation of LDL were also increased in HOME cells treated with TNF-alpha or IL-1. TNF-alpha-induced enhancement of LDL receptor gene expression was not observed when cycloheximide was present. Cellular mRNA level of SP-1 gene was increased about 3-4-fold at 12 h after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of LDL receptor gene as well as SP-1 gene by TNF-alpha. Gel retardation assay with the SP-1 consensus fragment showed that SP-1 binding activity was increased about 4-5-fold 12-24 h after treatment with TNF-alpha. NF-kB binding activity was also dramatically increased, but there is no NF-kB motif on the promoter for LDL receptor gene. The induction of LDL receptor by TNF might be mediated through a transcription factor, SP-1.