Biomedical subjects

Martina Schnölzer

Publications and source records attributed to Martina Schnölzer.

At least 19 recordsLinked to original sources

PubMed2006

Expression of GAGE family proteins in malignant melanoma.

Cancer/testis antigens are considered as promising targets for immunotherapy against tumors including malignant melanoma. One group of these antigens is the GAGE antigen family. In this study, we obtained recombinant GAGE-7b protein against which a rabbit antiserum was generated. The polyclonal, monospecific antibodies were used to analyze the expression of GAGE family proteins in human melanoma tissues and cell lines. GAGE expression in melanoma cell lines ranged from 41% to 58% and in melanoma tissues from 22% to 53%. Immunohistochemical analysis of melanoma tumors revealed a rather heterogeneous expression of GAGE resulting in individual positive cells or foci of stained cells. Furthermore, we could show that autoantibodies against GAGE family proteins are detectable in 6% of melanoma patients. Besides, we first demonstrated that the expression of GAGE family proteins can be stimulated with 5'-aza-2'-deoxycytidine and trichostatin A. Through upregulation of protein expression GAGE family proteins might develop into promising targets for immunotherapy of melanoma and other tumors.

Antigens, Neoplasm↗

PubMed2006

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B(2) from higher vertebrates.

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.

Amino Acid Sequence↗

PubMed2006

Human SGT interacts with Bag-6/Bat-3/Scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest.

The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43-57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression.

Apoptosis↗

PubMed2006

Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules.

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.

Caco-2 Cells↗

PubMed2006

Characterization of a hepatitis B and hepatitis delta virus receptor binding site.

Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion- and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) a myristoylated HBVpreS/2-48-GST fusion protein efficiently interferes with HBV infection of HepaRG cells; (2) deletions and point mutations in the highly conserved preS1 sequence between amino acids 11 through 21 result in the loss of infection inhibition activity; (3) hepatitis B viruses carrying single amino acid exchanges within this region lose infectivity; and (4) HDV infection of HepaRG cells can be inhibited by myristoylated HBVpreS peptides with the same specificity. In conclusion, HBV and HDV use at least one common step to enter hepatocytes and require a highly conserved preS1-sequence within the L-protein. This step is exceptionally sensitive toward inactivation by acylated HBVpreS1 peptides, which therefore represent a novel group of entry inhibitors that could be used for the treatment of hepatitis B and D.

Amino Acid Sequence↗

PubMed2005

Phosphorylation on Ser106 modulates the cellular functions of the SHOX homeodomain protein.

Mutations within the homeobox SHOX gene have been associated with short stature and the skeletal deformities found in Léri-Weill, Turner and Langer syndromes implying an involvement of SHOX in growth and bone formation. Despite its clinical significance, the precise role of SHOX and the mechanisms that modulate its functions remain unknown. We reported previously that SHOX is a nuclear protein that specifically binds DNA and acts as a transcriptional activator. We have shown that ectopic expression of SHOX leads to cell-cycle arrest and apoptosis in osteosarcoma and primary cells. To further characterize SHOX, we investigated whether the protein could be a target for phosphorylation. Here, we report that SHOX is phosphorylated exclusively on serine residues in vivo. Two-dimensional phospho-peptide mapping showed that SHOX is phosphorylated to various extents on multiple sites. Site-directed mutagenesis demonstrated that serine 106 is the major SHOX phosphorylation site. We show also that casein kinase II phosphorylates SHOX on serine 106 efficiently in vitro and specific casein kinase II inhibitors reduce SHOX phosphorylation strongly in vivo. Finally, we provide evidence that phosphorylation may play an important role in modulating SHOX biological activities, since a S106A SHOX mutant, defective in phosphorylation, does not activate transcription and fails to induce cell-cycle arrest and apoptosis.

Amino Acid Sequence↗

PubMed2005

The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7.

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.

Animals↗

PubMed2005

Xylosyltransferase I acceptor properties of fibroblast growth factor and its fragment bFGF (1-24).

Human basic fibroblast growth factor (bFGF) is a heparin-binding growth factor containing a G-S-G-motif which is a potential recognition sequence of xylosyltransferase I (XT-I). Here, we show that the recombinant human bFGF was xylosylated in vitro by human XT-I and that the fragment bFGF (1-24) is a good XT-I acceptor (K(m) = 20.8 microM for native XT-I and K(m) = 22.3 microM for recombinant XT-I). MALDI and MALDI-PSD time-of-flight mass spectrometric analyses of the xylosylated bFGF protein demonstrate the transfer of xylose to the serine residue of the G-S-G-motif in the amino terminal end of bFGF. The peptide bFGF (1-24) is well suitable as an acceptor substrate for XT-I and can be used in a radiochemical assay to measure the XT-I activity in cell culture supernatant and human body fluids, respectively. Furthermore, we could demonstrate that the XT-I interacts strongly with heparin and that this glycosaminoglycan is a predominantly non-competitive inhibitor of the enzyme using the fragment bFGF (1-24) as xylose acceptor.

Amino Acid Sequence↗

PubMed2005

Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin-3 and supports cell migration.

C4.4A is a member of the Ly6 family, with low homology to uPAR. It has been detected mainly on metastasizing carcinoma cells and proposed to be involved in wound healing. So far, C4.4A has been observed as an orphan receptor, and its functional activity has not been explored. Using recombinant rat C4.4A (rrC4.4A) made in a eukaryotic expression system, we demonstrate by immunohistology that C4.4A ligands are strongly expressed in tissues adjacent to squamous epithelia of, e.g., tongue and esophagus, the expression pattern partly overlapping with laminin (LN) and complementing the C4.4A expression that is found predominantly on the basal layers of squamous epithelium. ELISA screening of several components of the extracellular matrix revealed selective binding of rrC4.4A to LN1 and LN5 and that transfection of the BSp73AS tumor line with C4.4A cDNA (BSp73AS-1B1) promoted LN1 and LN5 binding. Binding of BSp73AS-1B1 to LN5 and, less markedly, LN1 induced spreading, lamellipodia formation and migration. C4.4A also associates with galectin-3 in nontransformed tissues and tumor lines. There is evidence that the association of C4.4A with galectin-3 influences LN adhesion. C4.4A was described originally as a metastasis-associated molecule. Our findings that LN1 and LN5 are C4.4A ligands, that galectin-3 associates with C4.4A and that C4.4A ligand binding confers a migratory phenotype are well in line with the supposed metastasis association.

Animals↗

PubMed2005

The tetraspanin D6.1A and its molecular partners on rat carcinoma cells.

Tetraspanins function as molecular organizers of multi-protein complexes by assembling primary complexes of a relatively low mass into extensive networks involved in cellular signalling. In this paper, we summarize our studies performed on the tetraspanin D6.1A/CO-029/TM4SF3 expressed by rat carcinoma cells. Primary complexes of D6.1A are almost indistinguishable from complexes isolated with anti-CD9 antibody. Indeed, both tetraspanins directly associate with each other and with a third tetraspanin, CD81. Moreover, FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1), an Ig superfamily member that has been described to interact with CD9 and CD81, is also a prominent element in D6.1A complexes. Primary complexes isolated with D6.1A-specific antibody are clearly different from complexes containing the tetraspanin CD151. CD151 is found to interact only with D6.1A if milder conditions, i.e. lysis with LubrolWX instead of Brij96, are applied to disrupt cellular membranes. CD151 probably mediates the interaction of D6.1A primary complexes with alpha3beta1 integrin. In addition, two other molecules were identified to be part of D6.1A complexes at this higher level of association: type II phosphatidylinositol 4-kinase and EpCAM, an epithelial marker protein overexpressed by many carcinomas. The characterization of the D6.1A core complex and additional more indirect interactions will help to elucidate the role in tumour progression and metastasis attributed to D6.1A.

Animals↗

PubMed2005

Decoding Tat: the biology of HIV Tat posttranslational modifications.

The Tat protein is a viral transactivator that activates HIV transcription through complex interactions with RNA and host cell factors. Tat undergoes multiple posttranslational modifications that regulate the dynamics and complexity of these interactions. The biology of these modifications and their role in Tat function are reviewed.

Arginine↗

PubMed2005

Probing lysine acetylation in proteins: strategies, limitations, and pitfalls of in vitro acetyltransferase assays.

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF).

Acetylation↗

PubMed2005

A proteomic investigation into etoposide chemo-resistance of neuroblastoma cell lines.

Neuroblastoma, one of the most common pediatric solid tumors, originates from the peripheral sympathetic nervous system and is responsible for approximately 15% of all childhood cancer deaths. Among the several antineoplastic drugs used in neuroblastoma chemotherapeutic protocols, topoisomerase inhibitors (i.e., etoposide) represent the most commonly used. Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs, mainly reducing the ability of neoplastic cells to start programmed cell death when exposed to antineoplastic drugs. The aim of this study was to determine, by means of proteomics, potential markers of etoposide resistance in human neuroblastoma cell lines as well as to investigate protein levels and modifications possibly involved in the onset of resistance. The etoposide resistant clone showed overexpression of the following proteins: peroxiredoxin 1, beta-galactoside soluble lectin binding protein, vimentin (three protein spots), heat shock 27 kDa protein (two protein spots) and heterogeneous nuclear ribonucleoprotein K. In addition, we also found down-regulation of dUTP pyrophosphatase. This investigation might represent a first step towards the development of novel prognostic markers of neuroblastoma chemotherapy.

Antineoplastic Agents, Phytogenic↗

PubMed2005

Large-scale protein expression for proteome research.

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.

Amino Acid Sequence↗

PubMed2005

Transcript and proteome analysis reveals reduced expression of calgranulins in head and neck squamous cell carcinoma.

The calcium-binding proteins of the S100 and the annexin protein families have been implicated in a variety of important physiological functions including membrane remodeling, calcium-related intracellular signaling, cytoskeleton dynamics, tissue homeostasis, and formation of the cornified envelope in differentiating keratinocytes. Deregulated expression of members of these families has been reported in different types of neoplasia and other diseases, but the results were not consistent. Here we have applied a combination of cDNA microarrays, quantitative reverse transcriptase-PCR (qRT-PCR) and surface enhanced laser desorption ionisation-time of flight mass spectrometry (SELDI-TOF MS) to study differential expression of these genes in head and neck squamous cell carcinoma (HNSCC). The calgranulins A and B and annexins 1 and 2 were found to be down-regulated in HNSCC, compared with normal mucosa, at both the mRNA and protein level. Upon validation of the differential gene expression by tissue microarray immunohistochemistry, we detected novel expression patterns of calgranulins A and B both in normal mucosa as well as in HNSCC. In contrast to squamous cancer of skin and other cancers in which the calgranulins were found to be up-regulated, most HNSCC showed reduced and widely deranged staining patterns including heterogeneous nuclear, cytoplasmic and membranous staining, and even enhanced staining in the tumor stroma. These observations suggest that the normal function of the calgranulins A and B in mucosa might be different from that in skin.

Calgranulin A↗

PubMed2005

Analysis of p300 acetyltransferase substrate specificity by MALDI TOF mass spectrometry.

Acetyltransferase enzymes target specific lysine residues in substrate proteins. While the list of histone and nonhistone substrates is growing, the mechanisms of substrate selection remain unclear. Here, we describe a mass spectrometric approach to examine the site selection of the acetyltransferase p300 in the HIV-1 protein Tat. Tat is acetylated by p300 at a single lysine (K50) within its basic RNA-binding domain. To determine the sequence requirements for K50 recognition within this domain, we synthesized mixtures of "degenerated" Tat peptides, in which one of the surrounding residues was substituted by all proteinogenic amino acids. Peptide mixtures were assembled based on nonoverlapping peptide masses and acetylated by p300 in a standard in vitro acetylation reaction. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified amino acid substitutions that prevented acetylation by p300. This approach represents a fast and comprehensive screening method that was applied to the six surrounding residues of K50 in Tat. It can be applied to any known acetyltransferase substrate and might help to define consensus recognition sequences for individual acetyltransferase enzymes.

Acetylation↗

PubMed2005

Rat tissue kallikrein releases a kallidin-like peptide from rat low-molecular-weight kininogen.

The kallikrein-kinin system is subdivided into the plasma and tissue-kallikrein-kinin system, with bradykinin (BK) and kallidin (KAL) (Lys(0)-bradykinin) as functional peptides. This occurs in both humans and other mammals. Both peptides are released by plasma and tissue-kallikrein. BK, but not KAL, has been detected in rats until now. One can explain this observation by the structural differences found in the sequence of rat high- and low-molecular kininogen containing an Arg-residue instead of a Lys-residue in front of the N-terminus of the BK sequence. Nevertheless, we were able to measure a kallidin-like peptide (KLP), in rat plasma and urine, using a specific KAL antiserum. In order to confirm our data, we isolated low-molecular-weight kininogen from rat plasma and incubated it with purified rat glandular kallikrein. The generated peptide was retained on a high-pressure liquid chromatography column and displaced by an excess of angiotensin I. The KLP-containing fraction was identified with the KLP radioimmunoassay. A specific ion signal with a mass to charge ratio (m/z) of 1216.73 was detected with matrix-assisted laser desorption/ionization mass spectrometry. As proposed earlier, the structure of this peptide is Arg(1)-KAL, instead of Lys(1)-KAL. The structural similarity between the Lys- and the Arg-residue explains the high crossreactivity (80%) of KLP with the specific KAL antibody. The incubation of KLP with angiotensin-converting enzyme yields two molecules with masses of 913.4 and 729.3 containing the sequence H-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-OH and H-Arg-Arg-Pro-Pro-Gly-Phe-OH. The enzymatic cleavage could be inhibited by captopril. The data suggest that in rats, as in other mammals, the tissue kallikrein-kinin system mediates its physiological effects via a kallidin-like peptide, which is Arg(1)-kallidin (Arg(0)-bradykinin).

Amino Acid Sequence↗

PubMed2004

CD44 variant isoforms associate with tetraspanins and EpCAM.

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-beta-cyclodextrin (MbetaCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell-matrix and cell-cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7-EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.

Adenocarcinoma↗