The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae.

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 A, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2-34, reveal an unprecedented mode of inhibitor-enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Cathepsin D from the liver of the antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures1.

Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion-exchange chromatography followed by affinity chromatography on concanavalin-A Sepharose. The purified enzyme showed a molecular mass of 40 kDa and displayed optimal activity at pH 3.0 with a synthetic chromogenic substrate. The N-terminal sequence of this proteinase was determined by automated Edman degradation and was used to design a primer for use in reverse-transcriptase polymerase chain reaction. The open reading frame of the cloned cDNA encoded an aspartic proteinase, which contained the experimentally determined N-terminal sequence. The predicted sequence (396 residues) had a high similarity with those of cathepsin D from various vertebrate sources, but was considerably different from that of nothepsin, a distinct aspartic proteinase described previously from Antarctic fish [1]. Determination of kinetic parameters for substrate hydrolysis showed that, at temperatures between 8 and 50 degrees C, the icefish cathepsin D had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50 degrees C and half-lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.

Cloning, expression and characterisation of murine procathepsin E.

The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli. The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors. The majority of the recombinant enzyme was present as a homodimer (Mr approximately 80) in which the two monomers were linked by an intermolecular disulfide bond. By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N-terminus of the mature proteinase. The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full-length cDNA now enables structural investigations and attempts to generate 'knock-out' mice deficient in this important aspartic proteinase to be undertaken.

Analysis of latent forms of renin using antibodies raised against the propart segment of human prorenin: validation with representative samples of ovarian cyst and follicular fluids.

Antisera were raised against synthetic peptides from the prosegment of human prorenin. The use of each of these for detection of the appropriate prosegment region of prorenin was validated by development of an ELISA protocol standardised with recombinant prorenin present in culture medium conditioned by myeloma cells transfected with a prorenin expression plasmid. Detection of the respective epitopes in the prosegment required prior exposure of the prorenin in the medium to acid pH in order to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. By these ELISA protocols, the form of latent renin present in representative samples from ovarian cyst and follicular fluids was analysed; one follicular cyst fluid was found to contain full-length prorenin whereas the fluid from a benign cyst and ovarian follicular fluid samples contained the precursor in truncated form.

Presence and possible significance of immunocytochemically demonstrable metallothionein over-expression in primary invasive ductal carcinoma of the breast.

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

Kinetic parameters for the generation of endothelins-1,-2 and -3 by human cathepsin E.

The specific conversion of human endothelin (ET) precursors big ET-1, big ET-2 and big ET-3 into their respective ET by cathepsin E was examined. Comparable pH optima were obtained for ET-1 and ET-2 generation, whereas effective conversion of big ET-3 into ET-3 necessitated a lower pH value. Determination of kinetic parameters (Km, kcat.) for all three conversions indicated that the precursors were efficiently bound by cathepsin E. The significance of the values obtained for the catalytic-centre activities and the effect of a specific inhibitor are discussed.

Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

Generation of human endothelin by cathepsin E.

Highly-purified cathepsin D processed human big endothelin1-38 into endothelin-like fragments but did not appear to generate endothelin1-21 under the conditions employed. By contrast, human cathepsin E specifically cleaved human big endothelin into endothelin1-21 and the C-terminal fragment under identical conditions but did not degrade either product further.

Immunological distinction between piscine and mammalian metallothioneins.

1. Separate antisera to metallothioneins (MT) from rainbow trout and horse were produced in mice and their reactivity with the respective immunogen was confirmed using an ELISA. 2. The ELISA, used in a competitive mode, revealed that the anti-horse MT serum did not cross-react with trout MT. Reciprocally, the anti-trout MT serum did not show any reactivity with horse MT. 3. The anti-rainbow trout MT serum was shown to cross-react totally with MTs from plaice, flounder, turbot, perch, salmon and pike, but exhibited no reactivity towards MTs from human, mouse, rat, worm or crab. Partial reactivity with the proteins isolated from oyster and mussel was demonstrated. 4. The anti-horse MT serum cross-reacted totally with MTs from human, rat and rabbit but no reactivity was demonstrable when MT from either plaice or worm was tested. 5. The behaviour of apo-, holo- and recombinant rainbow trout MTs, in which the metal content was different, indicated that reactivity with anti-MT antibodies was not dependent on the presence or nature of the metals bound in the protein. 6. The patterns of reactivities were analysed in relation to the known amino acid sequences of MT.