Bacterial community shift is induced by dynamic environmental parameters in a changing coastal ecosystem (northern Adriatic, northeastern Mediterranean Sea)--a 2-year time-series study.

The potential link between the microbial dynamics and the environmental parameters was investigated in a semi-enclosed and highly dynamic coastal system (Gulf of Trieste, northern Adriatic Sea, NE Mediterranean Sea). Our comprehensive 2-year time-series study showed that despite the shallowness of this area, there was a significant difference between the surface and the bottom bacterial community structure. The bottom bacterial community was more diverse than the surface one and influenced by sediment re-suspension. The surface seawater temperature had a profound effect on bacterial productivity, while the bacterial community structure was more affected by freshwater-borne nutrients and phytoplankton blooms. Phytoplankton blooms caused an increase of Gammaproteobacteria (Alteromonadaceae, SAR86 and Vibrionaceae) and shift in dominance from SAR11 to Rhodobacteraceae taxon at the surface. Our results propose the importance of the water mass movements as drivers of freshwater-borne nutrients and of allochthonous microbial taxa. This study emphasizes the prediction power based on association networks analyses that are fed with long-term measurements of microbial and environmental parameters. These interaction maps offer valuable insights into the response of marine ecosystem to climate- and anthropogenic-driven stressors.

Selective targeting of tumor and stromal cells by a nanocarrier system displaying lipidated cathepsin B inhibitor.

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L.

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.

Why some adults with intellectual disability consult their general practitioner more than others.

BACKGROUND: This research identifies factors affecting why some adults with intellectual disability (AWIDs) consult their general practitioner (GP) more than others. Little is known about these factors, despite AWIDs having higher health needs and reduced longevity. Current barriers to accessing health care need to be understood and overcome to achieve improved health outcomes. METHODS: A secondary analysis of data obtained from a stratified randomised sample of AWIDs participating in a cluster randomised trial of hand held health records. The number of GP consultations was obtained retrospectively for the year preceding initial health interviews from GP records. AWIDs and their carers were given separate health interviews using identical/adapted questions where possible. RESULTS: Two hundred and one AWIDs and or their carers from 40 practices participated (response rate 64.6%) with GP consultation data extracted for 187 AWIDs. Overall consulting levels were low, 3.2 per annum for women and 2.2 for men. Increased age, gender (women) and type of carer (paid) were all significantly associated with increased consultations. Carers reporting health problems, medications reported by AWIDs, medications recorded in GP records, and pain reported by AWIDs were also significant factors affecting consultations to GP practices after adjustment for age and type of carer. CONCLUSIONS: Overall consultation rates were lower than expected, and affected by age, gender and type of carer. Targeted interventions are needed to improve attendance and promote health.

Potential transfer of aquatic organisms via ballast water with a particular focus on harmful and non-indigenous species: A survey from Adriatic ports.

Ballast water discharges may cause negative impacts to aquatic ecosystems, human health and economic activities by the introduction of potentially harmful species. Fifty untreated ballast water tanks, ten in each port, were sampled in four Adriatic Italian ports and one Slovenian port. Salinity, temperature and fluorescence were measured on board. Faecal indicator bacteria (FIB), phyto- and zooplankton were qualitatively and quantitatively determined to identify the species assemblage arriving in ballast water. FIB exceeded the convention standard limits in 12% of the sampled tanks. Vibrio cholerae was not detected. The number of viable organisms in the size groups (minimum dimension) <50 and ≥10 µm and ≥50 µm resulted above the abundances required from the Ballast Water Management Convention in 55 and 86% of the samples, respectively. This is not surprising as unmanaged ballast waters were sampled. Some potentially toxic and non-indigenous species were observed in both phyto- and zooplankton assemblages.

Crystal structure of porcine cathepsin H determined at 2.1 A resolution: location of the mini-chain C-terminal carboxyl group defines cathepsin H aminopeptidase function.

BACKGROUND: Cathepsin H is a lysosomal cysteine protease, involved in intracellular protein degradation. It is the only known mono-aminopeptidase in the papain-like family and is reported to be involved in tumor metastasis. The cathepsin H structure was determined in order to investigate the structural basis for its aminopeptidase activity and thus to provide the basis for structure-based design of synthetic inhibitors. RESULTS: The crystal structure of native porcine cathepsin H was determined at 2.1 A resolution. The structure has the typical papain-family fold. The so-called mini-chain, the octapeptide EPQNCSAT, is attached via a disulfide bond to the body of the enzyme and bound in a narrowed active-site cleft, in the substrate-binding direction. The mini-chain fills the region that in related enzymes comprises the non-primed substrate-binding sites from S2 backwards. CONCLUSIONS: The crystal structure of cathepsin H reveals that the mini-chain has a definitive role in substrate recognition and that carbohydrate residues attached to the body of the enzyme are involved in positioning the mini-chain in the active-site cleft. Modeling of a substrate into the active-site cleft suggests that the negatively charged carboxyl group of the C terminus of the mini-chain acts as an anchor for the positively charged N-terminal amino group of a substrate. The observed displacements of the residues within the active-site cleft from their equivalent positions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the substrate, resulting in exopeptidase activity.

Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease.

BACKGROUND: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored. RESULTS: The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. CONCLUSIONS: The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases.

Lysosomal cysteine proteases: more than scavengers.

Lysosomal cysteine proteases were believed to be mainly involved in intracellular protein degradation. Under special conditions they have been found outside lysosomes resulting in pathological conditions. With the discovery of a series of new cathepsins with restricted tissue distributions, it has become evident that these enzymes must be involved in a range of specific cellular tasks much broader than as simple housekeeping enzymes. It is therefore timely to review and discuss the various physiological roles of mammalian lysosomal papain-like cysteine proteases as well as their mechanisms of action and the regulation of their activity.

Folding studies of the cysteine proteinase inhibitor--human stefin A.

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.

Refolding of recombinant sulphonated procathepsin S and of reduced chicken cystatin; implications for renaturation experiments.

Kinetic stopped-flow measurements of refolding of the recombinant sulphonated procathepsin S from 6 M urea are presented. The experiments were performed using intrinsic tryptophan fluorescence and fluorescence of the hydrophobic probe 1-anilino-naphthalene-8-sulfonate (ANS). Initially, (t1/2 = 3 +/- 1 ms) an intermediate with increased ANS fluorescence and protected tryptophan environment is formed. Much later, a slow increase in ANS fluorescence occurs with no accompanying changes in tryptophan fluorescence. The reaction of the slow ANS fluorescence increase correlates with the rate of aggregation as shown by the size exclusion chromatography (SEC). For comparison, the folding reactions of the reduced chicken cystatin were measured, both, by intrinsic tryptophan and extrinsic ANS fluorescence. An early intermediate forms very fast in the refolding of reduced chicken cystatin on 6-fold dilution from 5.7 M GuHCl (t1/2 = 5 +/- 2 ms), similarly to that observed for the sulphonated procathepsin S. ANS fluorescence and tryptophan fluorescence decrease further (t1/2 = 100 +/- 50 ms) leading to a late, 'more structured' intermediate which is prone to dimerization.

Compactness of the molten globule in comparison to unfolded states as observed by size-exclusion chromatography.

The volumes of elution of denatured states of four proteins at high urea (8 M) and ethylurea (6 M) concentration were determined. They were found equally unfolded in both solvents. The volumes of elution of the unfolded states were compared to those of the native states and of some molten globule intermediates. It has been shown that the protein proteinase inhibitor stefin B, exhibits 'molten globule'-like properties on acid denaturation. The high salt acidic intermediate (a molten globule) as well as the native state of stefin B eluted as dimers, at 18 degrees C. On thermal denaturation above 42 degrees C, the intermediate dissociated into compact monomers. The more stable stefin A, which is monomeric and does not transform into molten globule intermediates under similar perturbing conditions, was always used for comparison. The states of both, stefin A and B in 50% methanol were found to be monomeric and of native-like compactness.

Stoichiometry and heterogeneity of the pro-region chain in tetrameric human cathepsin C.

The subunit structure and composition of mature human cathepsin C, an oligomeric cysteine proteinase, has been characterised in detail. The heavy chain, light chain and pro-region peptides are shown to be held together solely by non-covalent interactions, and to be present in equimolar ratio, suggesting an important structural role for the residual pro-region chain which is strongly bound to the enzyme. The mass of the light chain, as determined by mass spectrometry, combined with its N-terminal sequence, determines the position of cleavage from the heavy chain. Amino-acid sequencing has led to definition of the 13.5 kDa N-terminal part of the pro-region which remains in the mature enzyme, the C-terminal moiety of 10 kDa being cleaved out and lost from the pro-peptide on activation. The residual pro-region is heterogeneous, a proportion being intact and the remainder being cleaved at alternative positions 58 or 61, yielding two smaller peptides joined by disulphide bond. The proportion of cleaved form was found to vary with tissue and enzyme preparation but did not affect enzyme activity. The molecular masses of the constituent chains after deglycosylation lead to a protein mass of 158 kDa. All four potential glycosylation sites are glycosylated.

Monoclonal antibodies to human stefin B and determination of their epitopes.

Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Crystallization of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data.

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.

Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen.

The structure of the wild-type human procathepsin B has been refined to a crystallographic R-value of 0.18 and R-free of 0.23 exploiting the data obtained from new crystals that diffract beyond 2.5 A resolution. The structure confirms two previously presented, lower-resolution structures. The structure of the propeptide chain folds on the surface of the enzyme domains and blocks access of substrate to the already formed active site. Abundant solvent molecules fill the cavities between the propeptide and the enzyme part of the molecule. The propeptide structure is compared with a substrate model in the S2, S1, S1' and S2' binding sites. In this crystal form the cathepsin B occluding loop residues adopt yet another conformation. The structures show that the occluding loop region between the residues Cys108 and Cys119 behaves quite independently from the rest of the structure and easily adapts to changes in environment. The variety of the observed conformations of the occluding loop is in agreement with other data showing that the loop is responsible for limiting cathepsin B activity to that of a carboxydipeptidase. The region before Cys108 is essentially the same as in the mature structure, whereas the region from Cys119 to Thr125 is raised compared to the mature form by the propeptide squeezed between it and the enzyme domains, surface. The structure strongly suggests that processing of procathepsin B during its autoactivation is not unimolecular.

The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution.

The solution structures of the phosphorylated form of native chicken cystatin and the recombinant variant AEF-S1M-M29I-M89L were determined by 2D, 3D and 4D-NMR. The structures turn out to be very similar, despite the substitutions and the phosphorylation of the wild-type. Their dominant feature is a five-stranded beta-sheet, which is wrapped around a five-turn alpha-helix, as shown by X-ray crystallographic studies of wild-type chicken cystatin. However, the NMR analysis shows that the second helix observed in the crystal is not present in solution. The phosphorylation occurs at S80, which is located in a flexible region. For this reason, very few effects on the structure are observed. Comparison of structures of the unphosphorylated variant and the wild-type shows small effects on H84 which is located in the supposed recognition site of the serine kinase. This recognition site appears to be well structured as a large loop-containing bulge of the beta-sheet. The N termini of both mutants, which contribute to a large extent to the binding to the proteinase, are very flexible. A loop structure involving the residues L7 to A10 as found in related inhibitors, such as in the kininogen domains 2 and 3, is not sufficiently populated to be observed.

Conformational variability of chicken cystatin. Comparison of structures determined by X-ray diffraction and NMR spectroscopy.

The structural model derived from X-ray crystallography for unphosphorylated wild-type chicken cystatin is compared with two chicken cystatin structures derived from NMR spectroscopy: the phosphorylated wild-type and the genetically engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall fold, but also significant differences in structurally variable segments of the polypeptide chain. The largest such segment, comprising residues 71 to 89, is a region characteristic of the family 2 cystatin inhibitors which contains a disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the accompanying article. In the crystal structure, the segment 71 to 76 is found as a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is completely undefined. The solution NMR structures on the other hand are disordered in the initial segment 72 to 80, have an extended conformation at 81 to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues 87 to 90. The segment comprising residues 53 to 57, with smaller variability, is of particular interest as the hairpin loop conserved throughout the cystatin superfamily which binds to the cysteine proteinase. In most of the solution NMR structures, this segment adopts a conformation more like that of stefin B, a family 1 cystatin inhibitor, as was observed in the crystal structure of its inhibitory complex with papain. The differences between the structures are rationalized by an examination of the crystal contacts generated by hypothetical crystal packing of the NMR structures. Additionally, the X-ray refinement shows evidence of conformational disorder in the crystal. Joint refinement with NOE restraints and reflection data does not produce a structure to satisfy the restraints of both methods.

The three-dimensional solution structure of human stefin A.

The three-dimensional solution structure of recombinant human stefin A has been determined by a simulated annealing protocol using a total of 1113 distance and angle constraints obtained from 1H and 15N HMR spectroscopy. The solution structure is represented by a family of 17 conformers with an average root-mean-square deviation relative to the mean structure of 0.44 A for backbone atoms and 0.94 A for all heavy atoms for the main body of the structure. The protein has a well-defined global fold consisting of five anti-parallel beta-strands wrapped around a central five-turn alpha-helix. There is considerable similarity between the structural features of free stefin A in solution and the X-ray structure of the homologous protein stefin B in its complex with papain, but there are also some important differences in the regions which are fundamental to proteinase binding. The differences consist primarily of two regions of high conformational heterogeneity in free stefin A which correspond in stefin B to two of the components of the tripartite wedge that docks into the active site of the target proteinase. These regions, which are shown to be mobile in solution, are the five N-terminal residues and the second binding loop. In the bound conformation of stefin B they form a turn and a short helix, respectively.

Immunochemistry of leucocyte intracellular proteinase inhibitors.

The antiserum was raised in rabbits against intracellular inhibitors I-1, I-2 and I-3 isolated from the soluble phase of disrupted pig peripheral leucocytes. It was demonstrated with double immunodiffusion and with immunoelectrophoresis that the isolated inhibitors with different biochemical characteristics are three different, specific and unrelated proteins. With the techniques used, it was clearly confirmed that the inhibitors were isolated in a pure form and that they are located in cytoplasm and nucleus. The suppression of inhibitors by antiinhibitors antibodies was also demonstrated.