Biomedical subjects

Sonja Vorderwülbecke

Publications and source records attributed to Sonja Vorderwülbecke.

6 recordsLinked to original sources

PubMed2006

New multi protein patterns differentiate liver fibrosis stages and hepatocellular carcinoma in chronic hepatitis C serum samples.

AIM: To identify a multi serum protein pattern as well as single protein markers using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) for detection and differentiation of liver fibrosis (F1-F2), liver cirrhosis (F4) and hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). METHODS: Serum samples of 39 patients with F1/F2 fibrosis, 44 patients with F4 fibrosis, 34 patients with HCC were applied to CM10 arrays and analyzed using the SELDI-TOF ProteinChip System (PBS-IIc; Ciphergen Biosystems) after anion-exchange fractionation. All patients had chronic hepatitis C and histologically confirmed fibrosis stage/HCC. Data were analyzed for protein patterns by multivariate statistical techniques and artificial neural networks. RESULTS: A 4 peptide/protein multimarker panel (7486, 12,843, 44,293 and 53,598 Da) correctly identified HCCs with a sensitivity of 100% and specificity of 85% in a two way-comparison of HCV-cirrhosis versus HCV-HCC training samples (AUROC 0.943). Sensitivity and specificity for identification of HCC were 68% and 80% for random test samples. Cirrhotic patients could be discriminated against patients with F1 or F2 fibrosis using a 5 peptide/protein multimarker pattern (2873, 6646, 7775, 10,525 and 67,867 Da) with a specificity of 100% and a sensitivity of 85% in training samples (AUROC 0.976) and a sensitivity and specificity of 80% and 67% for random test samples. Combination of the biomarker classifiers with APRI score and alfa-fetopotein (AFP) improved the diagnostic performance. The 6646 Da marker protein for liver fibrosis was identified as apolipoprotein C-I. CONCLUSION: SELDI-TOF-MS technology combined with protein pattern analysis seems a valuable approach for the identification of liver cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C. Most probably a combination of different serum markers will help to identify liver cirrhosis and early-stage hepatocellular carcinomas in the future.

Adult↗

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↗

PubMed2004

Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli.

The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli. TF unifies two functions in one to promote proper protein folding in vitro. First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding. Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding. Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center. Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro. In contrast, all other activities of TF F198A tested were comparable with wild type TF. TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in vitro, and associated with nascent polypeptides in an in vitro transcription/translation system. Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of DeltatigDeltadnaK cells and prevented global protein misfolding at temperatures between 20 and 34 degrees C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.

Cytosol↗

PubMed2003

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Chaperonin 60↗

PubMed2003

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation.

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.

Endopeptidase Clp↗

PubMed2002

L23 protein functions as a chaperone docking site on the ribosome.

During translation, the first encounter of nascent polypeptides is with the ribosome-associated chaperones that assist the folding process--a principle that seems to be conserved in evolution. In Escherichia coli, the ribosome-bound Trigger Factor chaperones the folding of cytosolic proteins by interacting with nascent polypeptides. Here we identify a ribosome-binding motif in the amino-terminal domain of Trigger Factor. We also show the formation of crosslinked products between Trigger Factor and two adjacent ribosomal proteins, L23 and L29, which are located at the exit of the peptide tunnel in the ribosome. L23 is essential for the growth of E. coli and the association of Trigger Factor with the ribosome, whereas L29 is dispensable in both processes. Mutation of an exposed glutamate in L23 prevents Trigger Factor from interacting with ribosomes and nascent chains, and causes protein aggregation and conditional lethality in cells that lack the protein repair function of the DnaK chaperone. Purified L23 also interacts specifically with Trigger Factor in vitro. We conclude that essential L23 provides a chaperone docking site on ribosomes that directly links protein biosynthesis with chaperone-assisted protein folding.

Amino Acid Motifs↗