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Human cystatin C (hCC) is a small protein belonging to the cystatin family of papain-like cysteine proteinase inhibitors. We review the recent literature concerning structural aspects of hCC related to disease. We focus on the mechanisms of hCC dimerization, oligomerization, and amyloid formation. Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. hCC is one of the second-wave proteins that have been found to undergo amyloidosis associated with disease. For hCC, this includes cerebral amyloid angiopathy, as well as a disorder resulting in reduced male fertility.

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the Ki values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.

Stefin B (cystatin B) is an inhibitor of lysosomal cysteine cathepsins and does not inhibit cathepsin D, E (aspartic) or cathepsin G (serine) proteinases. In this study, we have investigated apoptosis triggered by camptothecin, staurosporin (STS), and anti-CD95 monoclonal antibody in the thymocytes from the stefin B-deficient mice and wild-type mice. We have observed increased sensibility to STS-induced apoptosis in the thymocytes of stefin B-deficient mice. Pretreatment of cells with pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited phosphatidylserine externalization and caspase activation, while treatment with inhibitor of calpains- and papain-like cathepsins (2S,3S)-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation nor phosphatidylserine exposure. We conclude that sensitization to apoptosis induced by STS in thymocytes of stefin B-deficient and wild-type mice is not dependent on cathepsin inhibition by stefin B.

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped-flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (kass = 5.8 x 10(7) M-1.s-1, kdiss = 4.9 x 10(-4) s-1 at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentrations it was pseudo-irreversible (kass = 6.14 x 10(7) M-1.s-1). The complex was formed directly lacking the fast pre-equilibrium step with the dissociation equilibrium constant of approximately 8 pM. The competitive nature of inhibition was confirmed. The kass was found to be pH-independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pKi = 5.2, pK2 = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pKi = 4.4, pK2 = 7.8).

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 degrees C, and it exhibited a prolonged lag phase at 30 degrees C and 37 degrees C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.

A new stefin B-type low-Mr, CPI was isolated from bovine thymus and subjected to structural analysis. The inhibitor consisted of 98 amino acids and its Mr was calculated to be 11,178. The NH2-terminal amino acid residue was blocked. The sequence was determined by automated sequencing of peptides derived by cleavage with cyanogen bromide and fragments of the inhibitor resulting from enzymatic digestion with β-trypsin and Staphylococcus aureus V-8 proteinase. The NH2-terminal blocking group was established with mass spectrometry. The inhibitor exhibits considerable sequence homology with inhibitors from the stefin family. Furthermore, a highly conserved QVVAG region within the stefin family is for the first time replaced by the QLVAG sequence.

A new stefin type low- M r , cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M r , was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S ( k ass ⩾ 1 × 10 6 M −1 · s −1 ) and forms very tight complexes with these enzymes ( K i , ⩽ 190 pM). The affinity for cathepsins B and H ( K i ⩾ 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

Recombinant cystatin C producing clones were isolated from a human placenta λgt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with β-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.

A model for the evolution of mammalian cysteine proteinase inhibitors has been constructed on the basis of sequence homology. This model suggests that the diversity of cysteine proteinase inhibitors has evolved from two ancestral units forming the building blocks of stefin and cystatin. Gene triplication of the archetypal inhibitor generated the kininogen heavy chain which contains three cystatin-like copies. Hence, the superfamily of mammalian cysteine proteinase inhibitors is constituted by at least three distinct families, with stefin, cystatin and kininogen as their prototypes.

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxy-nucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.

Stefin A, a cysteine proteinase inhibitor of the cystatin superfamily, has been found to be most abundant in epidermal cells. In order to determine its cellular function, we have expressed human stefin A in Escherichia coli using plasmid expression vectors under the control of bacteriophage T7 RNA polymerase. The heat-stable, antibody-positive bacterial product was isolated using a papain-Sepharose affinity column and was shown to inhibit two cysteine proteinases, papain and human cathepsin B. Recombinant stefin A may have commercial and therapeutic potential in situations requiring inhibition of cysteine proteinase activities, and in cosmetics, as an ingredient in skin creams. Gene expression system; DNA, recombinant; Stefin A; Cystatin A

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.

We report that a mutant of human stefin B is in a molten globule conformation. It has all the spectroscopic characteristics for such a state. We also demonstrate that the molten globule is oligomeric, eluting on SEC within a similar MW range than the higher order oligomers of the wild type protein, which is confirmed by DLS and AFM. Both, the higher oligomers and the molten globule state bind ANS, implying a high degree of hydrophobic patches exposure and partial opening of the structure. Finally, we demonstrate that the oligomeric molten globule is as toxic as the prefibrillar aggregates obtained at acid pH or the higher order oligomers prepared at neutral pH.

Innate immune responses are tightly regulated to avoid excessive activation and subsequent inflammatory damage to the host, and interleukin-10 (IL-10) plays a crucial role in preventing inflammation. Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases. In stefin B-deficient bone marrow-derived macrophages (BMDMs), we detected an increase in the induction of the LPS-induced pro-inflammatory signal nitric oxide (NO) but decreased IL-10 expression. The phosphorylation of ERK and p38 MAP-kinases was significantly decreased in stefin B-deficient macrophages, as was STAT-3 phosphorylation. These findings show that stefin B influences the expression of anti-inflammatory IL-10 in response to the TLR4 agonist LPS.

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1–112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113–225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.