Biomedical subjects

A Sickmann

Publications and source records attributed to A Sickmann.

13 recordsLinked to original sources

PubMed2007

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G(2)/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60-90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase.

BACKGROUND: Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens. METHODS: Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients' sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients. RESULTS: Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients' sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients' sera. CONCLUSIONS: Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust.

Adult↗

PubMed2001

Identification of post-translationally modified proteins in proteome studies.

Proteome studies are powerful tools to solve many different problems in metabolism, signal transduction, drug discovery, and other areas of interest in life sciences. Up to now, high-sensitive methods for protein identification after two-dimensional gel electrophoresis using mass spectrometry are available. However, the identification of post-translational modifications after two-dimensional gel electrophoresis is still an unsolved problem. In this paper, we want to give several examples for the successful identification of post-translational modifications and point mutations.

Animals↗

PubMed2001

Investigation of charge variants of rViscumin by two-dimensional gel electrophoresis and mass spectrometry.

A method for the analysis of the rViscumin heterodimer (recombinant mistletoe lectin) based on two-dimensional (2-D) polyacrylamide gel electrophoresis and mass spectrometry was developed and used for quality control concerning purity and homogeneity of the recombinant protein processed under Good Manufacturing Practice (GMP) conditions. A series of spots with different pI-values in the pH-gradient of both rViscumin A- and B-chain were observed independently from the experimental conditions like urea concentration, heat treatment or the use of cysteine alkylating agents. Comparative studies of the major spots using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography-electrospray ionization (LC-ESI)-MS and LC-ESI-tandem MS (MS/MS) after tryptic in-gel digestion resulted in a sequence coverage of 92% for the A-chain and 95% for the B-chain. No molecular differences like common chemical or post-translational modifications or nonenzymatic deamidation were found to cause the different charge values of the separated spots. Therefore, these protein spots were extracted from the 2-D gel and separated again by 2-D gel electrophoresis (termed Re-2-DE). Each of the single spots tested in the Re-2-DE experiment split up in the same heterogeneous pattern concerning the pI-values. We suggest that the observed charge variants of rViscumin are the result of conformational protein variants, existing in an equilibrium during sample preparation and/or isoelectric focusing and are not caused from microheterogeneity in the primary structure of rViscumin.

Amino Acid Sequence↗

PubMed2001

Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry.

The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom. Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis. Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes.

Amino Acid Sequence↗

PubMed2001

Phosphoamino acid analysis.

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. Often, phosphorylation acts as a molecular switch controlling the protein activity in different pathways as in metabolism, signal transduction, cell division etc. Therefore, identification of phosphoamino acids in proteins is an important task in protein analysis. Since the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) a separation of phosphorylated and dephosphorylated protein species is possible. The identification of phosphorylation sites from preparative 2-D gels need very sensitive mass spectrometry methods and a specific enrichment of the phosphoprotein or -peptide.

Amino Acid Sequence↗

PubMed2001

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.

1-Phosphatidylinositol 4-Kinase↗

PubMed2001

Conformational switch and role of phosphorylation in PAK activation.

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.

Animals↗

PubMed2001

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors.

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

Fungal Proteins↗

PubMed2000

Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.

Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca(2+)/calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca(2+) levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor -II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser(1177), as well as Ser(633) which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr(495) in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-III in vitro caused a highly reproducible partial (10-20%) NOS-III activation which was independent of Ca(2+)/CaM, and as much as a 4-fold increase in V(max) in the presence of Ca(2+)/CaM. cAK stimulation in intact endothelial cells also increased both Ca(2+/)CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca(2+)/CaM-independent and -dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I(2) pathways and a positive feedback mechanism for NO/cGMP signaling.

Amino Acid Sequence↗

PubMed2000

Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis.

The aim of this work is to display the protein composition of the cerebrospinal fluid by two-dimensional (2-D) gel electrophoresis and identify it using different mass spectrometric techniques. This will enable us to present an overview of the proteins in human cerebrospinal fluid. The comparison of 2-D gels will help us to analyze the normal protein variability in healthy persons and specific protein variations in patients with different neurological diseases (e.g., morbus Alzheimer, chorea Huntington). However, it is not possible to carry out 2-D gel electrophoresis directly with human cerebrospinal fluid due to the high amount of salts, sugars and lipids present. In addition, the total amount of protein is only as high as 0.3-0.7 microg/microL. Therefore, concentration and desalting steps using precipitation and ultrafiltration are necessary. To date we have been able to identify more than 65 spots from 2-D gels using matrix assisted laser desorption/ionization-mass spectrometry and electrospray ionization-mass spectrometry.

Amino Acid Sequence↗

PubMed2000

Identification of major histocompatibility complex class II-associated peptides derived from freshly prepared rat Langerhans cells using MALDI-PSD and Edman degradation.

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at -80 degrees C to prevent peptide losses.

Animals↗

PubMed2000

The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.). Integration of a prokaryotic core into a larger complex with organelle-specific functions.

We previously identified two multisubunit plastid RNA polymerases termed A and B. The B enzyme has a bacterial-type polypeptide composition and is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif); the A enzyme has a more complex subunit structure and is Rif-resistant. Here we report results of N-terminal sequencing and MS carried out with the A enzyme, which establish that the latter contains rpo gene products and is structurally related to the B enzyme. Furthermore, evidence is provided that the A enzyme can be converted into a Rif-sensitive enzyme form in a phosphorylation-dependent manner in vitro by a treatment that results in depletion of a beta-like subunit. Database searches using sequence information derived from additional polypeptides that are present in purified A preparations revealed sequence similarity with chloroplast proteins involved in RNA processing and redox control. This proteomics approach thus points to the complexity of the chloroplast transcription apparatus and its interconnections with post-transcriptional and signalling mechanisms.

Alkaline Phosphatase↗