Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.

The most frequently used method for establishing epidemiological relationships between Plesiomonas shigelloides strains is O:H serotyping. However, a number of strains are not serotypeable and isolates from diverse sources can display the same serovar. Moreover, since the zoonotic nature of Plesiomonas has been suggested and this hypothesis is based on the identical serovars found in animals and humans, we intend to use four DNA-based techniques: random amplified polymorphic DNA-PCR, enterobacterial repetitive intergenic consensus-PCR, repetitive extragenic palindromic-PCR, and pulsed field gel electrophoresis in order to screen 24 strains belonging to nine O:H serovars isolated from humans, animals, and the environment. In general, P. shigelloides showed a high genetic heterogeneity. Three pairs of strains, each containing a human and an animal isolate, displayed similar genotypes. This is the first report that provides molecular evidence that P. shigelloides may be zoonotic.

The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based sitedirected mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by 300-fold, >10-fold and 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one aminoacid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, 45% of the total binding energy, for inhibition of cathepsin B.Keywords: cathepsins; cystatin; cysteine proteases; papain; second binding loop.Cystatins are effective protein inhibitors of cysteine proteases of the papain superfamily (reviewed in [1][2][3][4]). Found both intracellularly and extracellularly, they are believed to control the activity of normal endogenous proteases, as well as to protect organisms from the harmful activity of exogenous cysteine proteases [1,[4][5][6][7][8][9][10][11]. They are generally classified into three families according to their size and the presence of internal disulfide bonds. Cystatins of family 1, also called stefins, are small nonglycosylated proteins 11-12 kDa in size without disulfide bonds. Family 2 cystatins are somewhat larger, 12-14 kDa, with a structure stabilized by two disulfide bonds. Kininogens, representing the third family, are glycosylated proteins of about 50-90 kDa.The single polypeptide chain of a kininogen contains three domains resembling family 2 cystatins.Cystatins competitively inhibit the activity of papainlike cysteine proteases by binding to the active site of the latter and forming a tight, reversible protein-protein complex. A model of the inhibition was initially proposed from computer docking experiments based on the X-ray structures of papain and chicken cystatin, a family 2 member [12]. This model was later substantiated by the X-ray structure of a complex of the family 1 cystatin, human cystatin B (stefin B), with papain [13], the only structure of a cystatin-protease complex determined so far. The N-terminal segment and two hairpin loops of the cys...

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.