Increase Of Substrate Hydrolysis Research Articles

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural “key code” present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100 280–288 by fibroblast-expressed enzymes

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100 280–288, YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.

Angiotensin-converting enzyme activity: a novel way of assessing pulmonary changes during total knee arthroplasty.

Emboli after tourniquet release (TR) during total knee arthroplasty (TKA) occur in all patients. This may lead to fat embolism syndrome with lung injury. Angiotensin-converting enzyme (ACE) lines the pulmonary endothelium, and a decrease in ACE metabolism or hydrolysis of (3)HBPAP ((3)H-benzoyl-Phe-Ala-Pro; a substrate specific for ACE) has been associated with lung injury. We evaluated the association of this assay with pulmonary changes during TKA. Eleven consecutive patients undergoing bilateral TKA had the ACE assay performed perioperatively. We determined substrate hydrolysis and pulmonary capillary surface area (capillary perfusion index; CPI) and correlated it with pulmonary vascular resistance (PVR) and clinical outcome. Ten of the 11 patients demonstrated an increase in substrate hydrolysis and CPI along with a decrease in PVR after first or second TR when compared with baseline values (P < 0.05). In the other patient, PVR continued to increase even after TR, whereas CPI and substrate hydrolysis decreased after surgery. Whereas all others did well clinically, this patient developed confusion and hypoxemia. In previous studies, a decrease in PVR with an increase in CPI, as exhibited by the 10 patients, has been associated with pulmonary capillary recruitment. We believe this to be an important mechanism by which the lungs are able to accommodate the burden of emboli at the time of TR.

Requirements for distinct steps of phospholipase Cgamma2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling.

Studies of PLCgamma (phospholipase Cgamma) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCgamma2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore, we have defined the roles of different domains of PLCgamma2 and, using a panel of cell lines deficient in components linked to PLCgamma2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCgamma2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCgamma2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr753-->Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr753/Tyr759 were essential, whereas the PLCgamma2 SH2 domains did not have an important role in the transient activation of Lyn-PLCgamma2 but may serve to stabilize an activated form in sustained activation.

Characterization of the Functional Interaction of Adipocyte Lipid-binding Protein with Hormone-sensitive Lipase

Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific protein-protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.

Evidence for the presence of protease inhibitors in eastern ( Crassostrea virginica) and Pacific ( Crassostrea gigas) oysters

The plasma of eastern ( Crassostrea virginica) and Pacific ( Crassostrea gigas) oysters were compared for levels of inhibitory activities against a variety of proteases. Representatives of the serine, cysteine, metallo and aspartic protease mechanistic classes were analyzed, including extracellular proteases produced by two oyster-associated pathogens; Perkinsus marinus and Vibrio vulnificus. In comparison to C. virginica, C. gigas plasma exhibited significantly higher specific inhibition levels (ng protease inhibited/ μg plasma protein) for papain ( P<0.001), pepsin ( P<0.001), P. marinus protease ( P<0.001), trypsin ( P=0.015), and V. vulnificus protease ( P<0.001). Plasma of C. gigas did not inhibit the metalloprotease thermolysin. Instead, a significant increase in substrate hydrolysis was seen in wells containing plasma and thermolysin in comparison to wells containing thermolysin only. A similar trend was noted for thermolysin with the eastern oyster samples. These studies indicate the presence of protease inhibitors in the plasma of Crassostrea spp., which may have an impact upon host defense mechanisms, in addition to other physiological roles.

Group IV Cytosolic Phospholipase A2 Binds with High Affinity and Specificity to Phosphatidylinositol 4,5-Bisphosphate Resulting in Dramatic Increases in Activity

The group IV cytosolic phospholipase A2 (cPLA2) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at physiologically relevant concentrations. Specifically, the presence of 1 mol% PtdIns(4,5)P2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA2. This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P2 binds to cPLA2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P2 on the activity of cPLA2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA2 with PtdIns(4,5)P2 and the interaction of the pleckstrin homology domain of phospholipase C delta 1 with PtdIns(4,5)2 combined with sequence analysis of cPLA2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA2. We further show that the very nature of the interaction of proteins such as cPLA2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.