A preliminary study of the activation of endogenous pancreatic exocrine enzymes during automated porcine islet isolation.

The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model. Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.

A Homogenous 384-Well High Throughput Screen for Novel Tumor Necrosis Factor Receptor: Ligand Interactions Using Time Resolved Energy Transfer.

The herpes virus entry mediator (HVEM) receptor and its ligand, HVEM-L, are involved in both herpes simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2) infection, and in T-cell activation such that antagonists of this interaction are expected to have utility in viral and inflammatory diseases. In this report we describe the configuration of a homogeneous 384-well assay based on time-resolved energy transfer from a europium chelate on the HVEM receptor to an allophycocyanin (APC) acceptor on the ligand. Specific time resolved emission from the acceptor is observed on receptor:ligand complex formation. The results of various direct and indirect labeling strategies are described. Several assay optimization experiments were necessary to obtain an assay that was robust to automation and file compound interference while sensitive to the effect of potential inhibitors. The signal was stable for more than 24 h at room temperature using the Eu(3+) chelates, suggesting no dissociation of the lanthanide ion. The 384-well assay was readily automated and was able to identify more than 99.5% of known positive controls in the validation studies successfully. Screening identified both a series of known potent inhibitors and several structural classes of hits that readily deconvoluted to yield single compound inhibitors with the desired functional activity in secondary biological assays. The equivalence of the data in 384- and 1536-well formats indicates that routine implementation of 1536-well chelate-based energy transfer screening appears to be primarily limited by liquid handling rather than detection issues.

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening.

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5'-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.

Maturation of Qa-1b class I molecules requires beta 2-microglobulin but is TAP independent.

Two conformationally distinct and stable forms of Qa-1b, one strongly associated with beta 2-microglobulin (beta 2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable beta 2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without beta 2m, there was no maturation of either form in cells from beta 2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires beta 2m for an initial folding step. However, beta 2m is not essential for subsequent processing of Qa-1b molecules.

Antigen processing by proteasomes: insights into the molecular basis of crypticity.

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation.

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.

A viral inhibitor of peptide transporters for antigen presentation.

Cytotoxic T lymphocytes lyse target cells after T-cell-receptor-mediated recognition of class I major histocompatibility complex molecules presenting peptides. Antigenic peptides are generated in the cytoplasm by proteasomes and translocated into the lumen of the endoplasmic reticulum (ER) by peptide transporters (TAP). Herpes simplex virus (HSV) expresses a cytoplasmic protein, ICP47, which seems to interfere with such immune surveillance by mediating retention of 'empty' class I molecules in the ER. By expressing ICP47 in HeLa cells under an inducible promoter, we show that ICP47 efficiently inhibits peptide transport across the ER membrane such that nascent class I molecules fail to acquire antigenic peptides. This inhibition was overcome by transfecting murine TAP. Further, we demonstrate that ICP47 colocalizes and physically associates with TAP within the cell. Inhibition of peptide translocation by a viral protein indicates a previously undocumented potential mechanism for viral immune evasion.

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity.

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.

Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex.

The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.

Peptidylglutamyl-peptide hydrolase activity of the multicatalytic proteinase complex: evidence for a new high-affinity site, analysis of cooperative kinetics, and the effect of manganese ions.

The multicatalytic proteinase (MCP) complex or proteasome is a major nonlysosomal proteinase of eukaryotic cells. The proteinase can cleave peptide bonds on the carboxyl side of hydrophobic, basic, or acidic amino acid residues. These activities have been referred to as "chymotrypsin-like", "trypsin-like", and "peptidylglutamyl-peptide hydrolase" activities, respectively, and have been shown to be catalyzed at distinct sites. The latter activity is often assayed with the synthetic peptide substrate Z-Leu-Leu-Glu-beta-naphthylamide (LLE-NA). N-tBoc-Ala-Ala-Asp-SBzl is also a substrate for the rat liver MCP, suggesting a broader specificity for cleavage on the carboxyl side of acidic residues than the peptidylglutamyl-peptide hydrolase activity previously reported. The pH optimum is in the range of pH 7.0-7.5. Studies of the dependence of velocity on LLE-NA concentration show (a) that there is a high-affinity site (LLE1) which obeys Michaelis-Menten kinetics with a Km value of approximately 100 microM and (b) that at higher substrate concentrations (LLE2) the curve is sigmoidal, suggesting either allosteric activation of the proteinase at a second site or the involvement of multiple catalytic sites which display positive cooperativity. Activity at the high-affinity site (LLE1) can be distinguished from that of the activity of the LLE2 component by the effect of inhibitors, divalent metal ions, and KCl, as well as by its response to heat treatment. The addition of 1 mM MnCl2 stimulates both LLE1 and LLE2 activities and also permits saturation of MCP with substrate at concentrations of LLE-NA below the solubility limit of this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)