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Papers by vito turk

Bba Proteins Proteomics, 2009

Biological Chemistry

ABSTRACT

Biological Chemistry, 2000

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly i... more Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.

bchm, 2007

Apoptosis is the major mechanism by which eukaryotic organisms eliminate potentially dangerous, s... more Apoptosis is the major mechanism by which eukaryotic organisms eliminate potentially dangerous, superfluous and damaged cells. Initially, nuclei and mitochondria were found to be the key organelles involved in the process. However, recent data suggest that lysosomes and the endoplasmic reticulum also play important roles in the process. A number of different stimuli were found to directly or indirectly target the lysosomal membrane, thereby inducing lysosomal permeabilization and the release of cysteine cathepsins and the aspartic protease cathepsin D into the cytosol. Once in the cytosol, cathepsins can trigger cell death by different mechanisms. Here we discuss the different signaling pathways used by lysosomal proteases to trigger apoptosis and their potential role in physiological processes.

Biological Chemistry, 1999

Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24... more Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24 kDa and an isoelectric point in the range of 8.2 to 8.6. The enzyme is inhibited by equistatin, which belongs to the thyropins, a new family of protein inhibitors, with an inhibition constant of Ki = 0.40 +/- 0.07 nM. Cruzipain, a cathepsin L-like enzyme sharing a 130 amino acid long C-terminal extension, is also strongly inhibited by equistatin (Ki = 0.028 +/- 0.006 nM). Together with previously reported data, these results further indicate that a functional heterogeneity exists among thyropin inhibitors, as demonstrated by their interaction with cathepsin S and cruzipain.

International Journal of Molecular Sciences, Oct 25, 2023

Biological Chemistry Hoppe-Seyler, 1987

Biochemical Journal, 1984

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the... more Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzym...

Zeitschrift für Naturforschung B, 1967

Advances in Experimental Medicine and Biology, 1995

Proteins which have the ability to inhibit proteolytic activity of certain enzymes are found thro... more Proteins which have the ability to inhibit proteolytic activity of certain enzymes are found throughout the plant kingdom. They are usually accumulated as storage proteins in seeds and tubers (1). Many inhibitors of serine (2–4), cysteine (5–8) but only a few inhibitors of aspartic (9) proteinases have been isolated from potato tubers. The protein sequences of two closely related isoinhibitors, PDI (10) and NID (11), isolated from potato tubers, were determined.

Frontiers in molecular neuroscience, 2012

Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus... more Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). Our previous results showed that thymocytes isolated from stefin B-deficient mice are more sensitive to apoptosis induced by the protein kinase C (PKC) inhibitor staurosporin (STS) than the wild-type control cells. We have also shown that the increased expression of stefin B in the nucleus of T98G astrocytoma cells delayed cell cycle progression through the S phase. In the present study we examined if the nuclear or cytosolic functions of stefin B are responsible for the accelerated induction of apoptosis observed in the cells from stefin B-deficient mice. We have shown that the overexpression of stefin B in the nucleus, but not in the cytosol of astrocytoma T98G cells, delayed caspase-3 and -7 activation. Pretreatment of cells with the pan-casp...

Biological Chemistry Hoppe-Seyler, 1989

Cell Death & Disease, 2013

European Journal of Biochemistry, 2000

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a mole... more Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of ≈ 33 kDa and pI 5.1–5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7–15.0 nm), but poorly or not at all by stefin B (Ki > 250 nm) and l‐kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA‐074 and GFG‐semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1′ position, although the enzyme cleaved all P1′ residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin ...

Proteins: Structure, Function, and Genetics, 1998

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow trac... more The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.