ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum.

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain. A highly positively charged nonapeptide within this domain is necessary and sufficient for binding to ribosomes. Binding of ERj1p to ribosomes involves the 28S ribosomal RNA and occurs at the tunnel exit. Additionally, ERj1p has a dual regulatory role in gene expression: ERj1p inhibits translation in the absence of BiP, and another charged oligopeptide within the cytosolic domain of ERj1p mediates binding of the nuclear import factor importin beta and import into the nucleus, thereby paving the way for subsequent action on genomic DNA.

BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP.

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.

p53 autoantibodies from patients with head and neck cancer recognise common epitopes on the polypeptide chain of p53.

In the present study, we analysed p53 autoantibodies from patients with head and neck cancer by ELISA, by Western blot using C- and N-terminal fragments of p53 and with peptide libraries of p53. We found that 8.2% of the patients with head and neck cancer developed antibodies against p53. Using additional p53 autoantibody-positive sera from patients with head and neck cancer, we found that all of these sera recognised common epitopes, which were also recognised by well-known mouse monoclonal antibodies against p53. Common epitopes are located in regions on the polypeptide chain of p53, which are functionally important for the role of p53 in growth control. Thus, the knowledge of these epitopes might be useful for the development of new strategies for cancer therapy.

MGEA6 is tumor-specific overexpressed and frequently recognized by patient-serum antibodies.

Tumorigenesis of meningioma has been associated with chromosome 22, most notably the NF2 gene, but additional genes have also been implicated in meningioma development. Previously, we have cloned the cDNAs for the meningioma expressed antigen 6 (MGEA6) and its splice variant MGEA11. Here, we show that antibodies against recombinantly expressed MGEA6/11 are found in 41.7% (10/24) of the sera from meningioma patients and in 2/8 sera of glioblastoma patients, whereas no response was seen in 12 sera from healthy persons. Western-blot analyses using generated polyclonal antibodies, revealed overexpression in meningioma and glioma tumor samples compared to normal brain. Immunohistochemical staining of tissue sections confirms reactivity in meningioma tumor cells and tumor cells of glial origin. We found no reactivity to normal astrocytes and only faint reactivity to normal leptomeninges. Sequence analysis predicted membranic localization of MGEA6/11, that was confirmed by cell fractionation. The immune response to MGEA6/11 is frequent in both meningioma and glioma patients and may likely be attributed to overexpression of the MGEA6/11 protein in the tumor cells.

The TRPV6 gene, cDNA and protein.

The mouse TRPV6 gene is localized on chromosome 6 and extends over 15.66kb. The encoded protein comprises 727 amino acid residues with a calculated relative molecular mass of 83,210Da. TRPV6 is glycosylated and both variants, the glycosylated and the de-glycosylated proteins, are recognized by various polyclonal and monoclonal antibodies, which were raised against TRPV6. Like human TRPV6, mouse TRPV6 binds calmodulin in the presence, but not in the absence of Ca2+. TRPV6 is abundantly expressed in mouse pancreas and placenta, and to a much lesser extend in mouse stomach and kidney. No transcript expression was detected in poly(A)+RNA isolated from heart, brain, intestine, esophagus or aortic endothelial cells.

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds.

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositide-dependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.

Prostacyclin in the cardiovascular system: new aspects and open questions.

Several indications exist that prostacyclin (PGI(2)) release in the cardiovascular system might be affected by cyclooxygenase (COX)-2-specific inhibitors. This could reflect an inhibition of PGI(2) synthesis in the endothelium although in these cells mainly COX-1 is expressed. Inflammation and stress induce COX-2 in smooth muscle cells which could have happened in patients with cardiac diseases. Herein, we show that also cardiomyocytes contain PGI(2) synthase in intercalated discs as a third source of PGI(2) in the cardiovascular system. Another aim of this study was to explain the finding that PGI(2) synthase in lipopolysaccharide (LPS)-treated smooth muscle cells, in contrast to endothelial cells, is resistant to nitration and inhibition by peroxynitrite. By using redox cyclers, the nitration occurred and confirmed our previous hypothesis that a high peroxidative activity of such cells keeps peroxynitrite below the effective levels of 50 nM. Considering enhanced oxidative stress in aged vessels, we postulated and verified that endothelial dysfunction in aged vessels is due to nitration and inhibition of PGI(2) synthase. Such data underline the role of PGI(2) as a potent mediator for regaining and maintaining the normal resting state of cells in a COX-2 dependent fashion.

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

Differential expression of electrogenic NBC1 (SLC4A4) variants in rat kidney and pancreas.

The purpose of this study was to determine expression and localization of NH(2)-terminal variants of the electrogenic Na(+)-HCO(3)(-) co-transporter NBC1 (SLC4A4) in the rat kidney and pancreas. We generated two anti-peptide antibodies: alpha333 against the "mste" start (kidney; kNBC1) and alpha332 against the "mede" start (pancreas; pNBC1). Transcripts for both NBC1 variants were detected in kidney and pancreas by RT-PCR, though kNBC1 was more prominent in the kidney and pNBC1 was more prominent in the pancreas. Similar protein expression levels were detected by immunoblotting of plasma membranes (PM) from kidney cortex and pancreas. Immunohistochemistry with alpha333 recognized the "mste"-epitope in the basolateral plasma membrane (BLM) of renal proximal tubule. The "mede"-protein (alpha332) was similarly localized although staining was much less and more diffuse. In the pancreas, alpha332 stained BLM of acinar and duct cells. Some isolated duct cells were also stained at the apical PM. The "mste"-protein (alpha333) was absent in acinar cells but was located at the apical PM of duct cells. The data indicate that the two NH(2)-terminal NBC1 variants are co-expressed in kidney and pancreas, where they may contribute to HCO(3)(-) transport and pH regulation.

Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p.

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins.

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.