The early history of intracellular cysteine proteinase inhibitors.

Scientists at the beginning of their careers often rely on the information obtained from review articles. Furthermore, the review articles are commonly used by sponsors in making their funding decisions of scientific projects. Thus it is very important to give a balanced view of the past as well as the present of the field discussed in those papers. In the present letter I wish to complement a review article previously published in Frontiers in Bioscience.

Epidermal SH-protease inhibitor (ACPI, cystatin A) in cancer. A short historical review.

This review article will re-report the findings from 30 years ago concerning the implications of the acid cystein proteinase inhibitor (ACPI) to the field of cancer research. The original article, published in 1980, has erroneously been omitted from electronic article databases. The findings reported in the original article suggest that some anaplastic lung cancers should be classified as epidermal. In this article we also consider the yet unknown physiological function of ACPI. Furthermore, we will address the simplification of the nomenclature of ACPI and related molecules.

Cystatin B as an intracellular modulator of bone resorption.

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.

Expression of cystatins, high molecular weight cytokeratin, and proliferation markers in prostatic adenocarcinoma and hyperplasia.

BACKGROUND: Prostatic adenocarcinoma is the most common malignancy among men in the western world but the diagnostic and prognostic criteria for it are still not clearly defined. Additional means for its diagnosis and prognosis are clearly needed. Previously it has been shown that cystatin A is expressed in the basal cells of normal prostate and the expression disappears in prostatic carcinoma. METHODS: We have now studied the expression of both cystatins A and B in benign prostatic hyperplasias (BPH), prostatic intraepithelial neoplasias (PIN) and carcinomas of the prostatic epithelium and compared it with the expression of high molecular weight (HMW) cytokeratin as well as the proliferation markers cyclin A and Ki-67. The expression of the proteins was immunohistochemically assessed using 33 total prostatectomy specimens. RESULTS: Cystatin A was expressed in the basal cells in all cases of BPH, low-grade PIN, and high-grade PIN whereas carcinomas showed no staining of cystatin A. The 34 beta E12 cytokeratin expression was similar to basal cystatin A staining and was not seen in carcinoma foci. Cystatin B showed both nuclear and cytoplasmic expression in the columnar epithelial cells. The decrease in median cytoplasmic staining of cystatin B in carcinomas compared to other lesions was significant, but there was a significant increase in expression with dedifferentiation of carcinoma. Also cyclin A and Ki-67 staining were significantly different in non-carcinomatous foci compared to carcinoma foci and had a remarkably similar negative correlations with basal cystatin A and 34 beta E12 staining. CONCLUSIONS: The results show that cystatin expression can be used as an aid in the diagnosis of prostatic adenocarcinoma and especially cystatin A in the distinction between high grade PIN and grade I carcinoma.

Immunolocalization of cysteine proteinases (cathepsins) and cysteine proteinase inhibitors (salarin and salmon kininogen) in Atlantic salmon, Salmo salar.

Tissue localization of cysteine proteinases (cathepsins) and their inhibitors (salarin, salmon kininogen) was performed in tissues of the Atlantic salmon. In skin, both epidermis and dermis were strongly stained by antisera against salarin and salmon kininogen. In epidermis the intercellular space seemed to be heavily stained (salarin). In kidney, the inhibitors were mainly localized to the interstitial capillaries. Also, some epithelial cells of the tubules (salarin) and some cells of the interstitium were stained. Mostly, the staining had a diffuse cytoplasmic localization. In the liver some hepatocytes were strongly positive for salarin and salmon kininogen. Purified fish cysteine proteinase inhibitors were not found to inhibit the growth of fish pathogenic bacteria and viruses. In the trunk kidney cathepsins B and L were localized in epithelial cells of the tubules (proximal part) and in cells of the interstitium. Mostly, the staining showed a prominent lysosomal localization. In head kidney large macrophage-like cells were positively stained for cathepsin B. The staining was localized to granula/vacuoles in the cytoplasm. In the liver, some hepatocytes were strongly stained and some were less strongly positive for cathepsin B and L. Mostly, the hepatocytes showed lysosomal staining. Cathepsin L was found in some big macrophage-like cells in the spleen. Mucosal epithelial cells of the esophagus and intestine seemed to be strongly stained for cathepsin B and L. The results show that cathepsins and their inhibitors are specifically and widely distributed in the Atlantic salmon skin indicating that they perform some biologically important and specific but so far unknown functions.

Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod.

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.