Biomedical subjects

Bernd Wiederanders

Publications and source records attributed to Bernd Wiederanders.

11 recordsLinked to original sources

PubMed2006

The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions.

The crystal structure of the active-site mutant Cys25 --> Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Angstrom resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S1' and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix alpha3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.

Amino Acid Sequence↗

PubMed2006

Microplate assay for quantitative determination of cathepsin activities in viable cells using derivatives of 4-methoxy-beta-naphthylamide.

A method is described allowing the selective determination of four cathepsins (B, H, K, and L) in live cells. Adherently growing cells are incubated with partially selective substrates for each cathepsin (peptidic derivatives of 4-methoxy-beta-naphthylamine) in microtiter plates together with nitrosalicylaldehyde. Using an appropriate reader accumulating fluorescent products may be detected continously or by end point measurement. Selectivity is achieved by running parallel assays containing inhibitors that are partially selective for each of the cathepsins (in case of cathepsin H, the nonlysosomal aminopeptidases are inhibited by bestatin). Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assay. The method was validated by measurements in cells isolated from cathepsin B(-/-)-, K(-/-)-, and L(-/-)- mice. This strategy suggests that the combination of two partially selective reaction partners, substrate and inhibitor can yield selective cathepsin assays.

2-Naphthylamine↗

PubMed2005

Leukemia inhibitory factor triggers activation of signal transducer and activator of transcription 3, proliferation, invasiveness, and altered protease expression in choriocarcinoma cells.

Extravillous trophoblast cells resemble cancer cells with regard to their intrinsic invasiveness. They invade decidual tissue, but, unlike tumor cells, shut down their invasive properties, when they become inappropriate. Stimuli involved in the modulation of invasion, as well as their underlying signaling mechanisms require further clarification. We were especially interested in discovering signals capable of stimulating invasion in otherwise low-invasive cells involved in reproduction. Using the choriocarcinoma cell line Jeg-3 as a model, we have addressed the potential role of cytokine/growth factor-driven activation of signal transducer and activator of transcription 3 (STAT3) in this process. Jeg-3 cells were treated with various factors known to induce trophoblast proliferation, differentiation, migration, or invasiveness (insulin-like-growth-factor-II (IGF-II), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF)). Only LIF elicited strong tyrosine phosphorylation and specific DNA-binding activity of STAT3. It induced a significant acceleration of cell proliferation and promoted the capability of Jeg-3 cells to invade into an artificial extracellular matrix. Moreover, LIF influenced the expression pattern of proteases and protease inhibitors with potential relevance for invasiveness (downregulation of mRNA for tissue inhibitor of metalloproteinase 1 (TIMP-1) and upregulation of mRNA for caspase-4). In conjunction with earlier work, in which we found that STAT3 DNA-binding activity was increased in invasive cells (choriocarcinoma, first trimester trophoblasts) and absent in non-invasive cells (term trophoblasts), these findings suggest a connection between LIF-driven STAT3 activity and invasiveness of choriocarcinoma and trophoblast cells.

Caspases↗

PubMed2004

Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis.

Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Real-time polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, real-time PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

Animals↗

PubMed2003

An unfolding/refolding step helps in the crystallization of a poorly soluble protein.

Proteins that are unstable or poorly soluble often elude crystallization. Here, a novel strategy is presented that leads to the crystallization of the isolated N-terminal propeptide of human procathepsin S, a proteinase belonging to the cathepsin L-like endopeptidases of the clan CA1 cysteine peptidases. Being very hydrophobic, the propeptide is extremely poorly soluble in aqueous solvents at neutral pH. Solubility is much better at acidic pH, but the native structure is destroyed under these conditions. A novel approach to the crystallization of this poorly soluble protein is presented in which it is first unfolded in an acidic buffer (pH 4.5) and then mixed with a nearly neutral crystallization buffer (pH 6.75) in which the native conformation should form spontaneously. Crystals were grown at a high concentration of MES (1.14 M) with 10% 2-propanol as precipitant. They belong to a tetragonal space group, with unit-cell parameters a = b = 151.1, c = 75.8 A. Diffraction data to a resolution of 3.5 A were obtained.

Cathepsins↗

PubMed2003

Functions of propeptide parts in cysteine proteases.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca(++) -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.

Amino Acid Sequence↗

PubMed2003

Structure-function relationships in class CA1 cysteine peptidase propeptides.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.

Cysteine Endopeptidases↗

PubMed2002

[What can experimental research offer to rheumatology today--the viewpoint of molecular biology? Contribution of molecular biology to pathogenesis research in rheumatology using the example of rheumatoid arthritis].

Molecular biology plays an increasing role for the development of innovative approaches to analyze the pathogenesis of rheumatic diseases and to improve diagnosis and therapy of these disorders. Some of these approaches/techniques have recently yielded important results, e.g. the analysis of 1) chromosomal aberrations (numerical and, in part, structural aberrations in synovial fibroblasts/macrophages from chronic joint inflammation); 2) cell clonality (oligoclonal expansion of synovial T-cells, B-cells, but also fibroblasts); 3) the importance of genetic factors (genome-wide screening for arthritis susceptibility genes); 4) mutations in key genes of cell cycle and/or function (mutations in p53 and proto-oncogenes in the inflamed synovial membrane); and 5) gene expression patterns (e.g. by high-density microarrays, custom arrays, in situ hybridization, and real-time PCR). It can be expected that these analyses will result in central new findings concerning the understanding of the pathogenetic basis of chronic inflammatory rheumatic diseases, with the potential to develop differential diagnostic criteria for these hitherto extremely heterogeneous diseases, and to create the basis for individual-oriented therapy.

Arthritis, Rheumatoid↗

PubMed2002

Foldase function of the cathepsin S proregion is strictly based upon its domain structure.

Folding of cathepsin S, like other cathepsin L-like proteases, depends on its proregion. The major part of the proregion forms a small domain distal from the catalytic centre, suggesting function(s) beyond active-site shielding. Using an optimised in vitro trans-refolding assay, we compared reactivation of denatured cathepsin S by the genuine propeptide, wild-type and ten selected mutants. Including structural data and binding constants, we identified the prodomain core and the hairpin region to be important for the foldase function.

Amino Acid Sequence↗

PubMed2002

Cytochemical demonstration of expression and distribution of non-glycosylated human lysosomal cathepsin S in HEK 293 cells.

The lysosomal cysteine protease cathepsin S is synthesized as inactive precursor at the rough endoplasmic reticulum (ER), further processed in the Golgi compartment and finally targeted to the lysosomes where it becomes activated by the proteolytic cleavage of the inhibitory propeptide. Biochemical studies with a non-glycosylated mutant of procathepsin S (plasma membrane binding at 2 degrees C, reuptake of secreted enzyme at 37 degrees C) led to the suggestion of an additional sorting motif in procathepsin S besides the classical Man-6-P recognition signal. In order to further confirm this suggestion on a morphological basis we performed a series of laser scanning confocal microscopy (CLSM) and electron microscopical analyses with HEK 293 cells expressing the mutant non-glycosylated procathepsin S. Immunolocalization with CLSM documented clearly a fine granular fluorescence in the paranuclear region of mutant expressing cells. Electron microscopy demonstrated the presence of cathepsin S immunoreactive deposits within cytosolic vacuoles (lysosomes), at the plasma membrane and in ER buds. These buds were also visible in the cytosol as well as in form of concentrated patches at the plasma membrane indicating the direct transport of (pro)cathepsin S from the ER to the cell surface.

Acid Phosphatase↗

PubMedDate not supplied

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain.

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.

Amino Acid Sequence↗