Kinetic analysis of the control through inhibition of autocatalytic zymogen activation.
Ontology highlight
ABSTRACT:
SUBMITTER: Manjabacas MC
PROVIDER: S-EPMC1130821 | biostudies-other | 1992 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundBefore furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.Methodology/principal findingsWe constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.Conclusions/significanceCollectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.
| S-EPMC2662429 | biostudies-literature
Project description:Protein phosphorylation and limited proteolysis are two most common regulatory mechanisms involving the energy-dependent covalent modification of regulatory enzymes. In addition to modifying other proteins, many protein kinases and proteases catalyse automodification reactions (i.e. reactions in which the kinase or zymogen serves as its own substrate), and their activities are frequently regulated by other regulatory ligands. In the present study, a kinetic analysis of autocatalytic reaction modulated by regulatory ligands is presented. On the basis of the kinetic equation, a novel procedure is developed to evaluate the kinetic parameters of the reaction. As an example of an application of this method, the effects of calcium ions on the autoacatalytic activation of trypsinogen by trypsin is re-examined. The results indicate that the binding affinity for Ca2+-bound trypsinogen to trypsin is at least two orders of magnitude higher than that for Ca2+-free trypsinogen, and therefore that the effect of Ca2+ ions on K(m*) values for trypsinogen is very much greater than that for the model peptides. Based on the experimental results, one possible molecular mechanism has been proposed.
| S-EPMC1224100 | biostudies-other
Project description:A global kinetic analysis of a general zymogen activation model, where not only the activating but also the activated enzyme suffer an irreversible inhibition is presented. A reaction in which the enzyme acts upon a substrate is coupled to monitor the process. In addition, we determined the corresponding kinetic equations for a number of particular cases of the general model studied. Finally, a kinetic data analysis and a procedure to discriminate among the different mechanisms considered, which are based on the kinetic equations obtained, are suggested.
| S-EPMC1132296 | biostudies-other
Project description:Inhibitor of apoptosis (IAP) proteins have a crucial role in apoptosis, through negative regulation of caspases in species from fruitflies to mammals. In Caenorhabditis elegans, however, no IAP homolog or caspase inhibitor has been identified, calling into question how the cell-killing caspase CED-3 can be negatively regulated. Here we show that inactivation of the C. elegans csp-3 gene, which encodes a protein similar to the small subunit of the CED-3 caspase, causes cells that normally live to undergo apoptosis in a CED-3-dependent manner. Biochemical analysis reveals that CSP-3 associates with the large subunit of the CED-3 zymogen and inhibits zymogen autoactivation. However, CSP-3 does not block CED-3 activation induced by CED-4, nor does it inhibit the activity of the activated CED-3 protease. Therefore CSP-3 uses a previously unreported mechanism to protect cells from apoptosis.
| S-EPMC2574878 | biostudies-literature
Project description:Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E*) to binding. Prothrombin exists mainly in the E* conformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E*-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis.
| S-EPMC5840343 | biostudies-literature
Project description:Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-filled cells of the interior. We also show that matriptase potently activates hepatocyte growth factor (HGF) in vitro, and that the HGF receptor, c-Met, is co-expressed in those cells that express activated matriptase. Our observations suggest that the matriptase-HGF-c-MET pathway has the potential to be engaged, primarily in proliferative cells rather than terminally differentiated epithelial cells of the human pilosebaceous unit.
| S-EPMC3873802 | biostudies-literature
Project description:Cells decide when, where, and to what level to express their genes by "computing" information from transcription factors (TFs) binding to regulatory DNA. How is the information contained in multiple TF-binding sites integrated to dictate the rate of transcription? The dominant conceptual and quantitative model is that TFs combinatorially recruit one another and RNA polymerase to the promoter by direct physical interactions. Here, we develop a quantitative framework to explore kinetic control, an alternative model in which combinatorial gene regulation can result from TFs working on different kinetic steps of the transcription cycle. Kinetic control can generate a wide range of analog and Boolean computations without requiring the input TFs to be simultaneously bound to regulatory DNA. We propose experiments that will illuminate the role of kinetic control in transcription and discuss implications for deciphering the cis-regulatory "code."
| S-EPMC5469051 | biostudies-literature
Project description:The migration of chemicals from polyurethane (PUR) is a concern in many applications, such as adhesives for food packaging. Low molecular weight catalysts, which are prone to migration, need to be eliminated from PUR, in particular those containing Sn or other metals. This is difficult partly due to many uncertainties of autocatalytic polyaddition between isocyanates and polyols. Hexamethylene and tolylene diisocyanates, HDI and TDI, are often reacted with macrodiols to produce prepolymers for PUR. This study measures isocyanate contents during the polyaddition of HDI and TDI with excess macrodiols. Ester-based macrodiols were reacted between 60 °C and 90 °C using 1 : 0.3 and 1 : 0.5 molar ratios to form OH-terminated prepolymers. Time-temperature superposition (TTS) was used to process the values of unreacted isocyanate fractions from several temperatures. Presumed activation energies and kinetic data scatter implied that polymerization has a distinct initial phase of conversion of unreacted diisocyanate into monofunctional adducts by addition to macrodiols, followed by more complex processes. Utilization of the activation energy from the initial phase and TTS application might allow the prediction of kinetic trends without the need for a large volume of accurate data. Such kinetic mapping should be useful for developing catalyst-free PUR with low levels of migrating chemicals.
| S-EPMC10037676 | biostudies-literature
Project description:The zymogen activation mechanism and physiological functions of the most ancient and highly conserved basic amino acid-specific proprotein convertase 7 (PC7) are not known. Herein, we characterized the biosynthesis, subcellular localization, and trafficking of the membrane-bound full-length rat and human PC7. The prosegment of PC7 is primarily secreted alone as a non-inhibitory protein via the conventional, Golgi-dependent, secretory pathway. Mature PC7 is partially sulfated and thus reaches the cell surface via the conventional route. However, a fraction of PC7 reaches the cell surface through a brefeldin A- and COPII-independent unconventional secretory pathway. The latter trafficking may explain the rapid (<10 min) transit of a fraction of PC7 from the ER to the cell surface. Electron microscopy further confirmed the localization of PC7 to the cell surface of HEK293 cells. Within the cytosolic tail, only two cysteines (Cys(699) and Cys(704)) are palmitoylated, but this modification does not affect the choice of trafficking pathway. Swapping the transmembrane-cytosolic tail (TMCT) sequences of the convertases Furin and PC7 revealed that PC7(TMCT-Furin) is much more sulfated and hence traffics more efficiently through the conventional secretory pathway. In contrast, the Furin(TMCT-PC7) is no longer sulfated and thus reaches the cell surface by the unconventional pathway. Because trafficking of PC7(CT-Furin) and Furin(CT-PC7) resemble their wild type counterparts, we deduce that the transmembrane domain of PC7 regulates the sorting of PC7 toward the unconventional secretory pathway. In conclusion, PC7 is distinct from other proprotein convertases in its zymogen activation, subcellular localization, and trafficking.
| S-EPMC3024769 | biostudies-literature
Project description:The global gene expression profiling of early T helper (Th) 1 and Th2 differentiation reveals that this process can be divided into two stages, activation and differentiation. The activation stage is manifested in coordinated mobilization of the replication machinery, a process that we hypothesize may be responsible for establishing genomewide opening of transcription loci. The molecular programs underlying the differentiation stage consist of highly regulated expression of functional groups of genes that are important for the biological properties of Th1/2 cells and transcription factors that are likely important in establishing terminal differentiation of these cells. The kinetics of expression pattern of a number of transcription factors shed new light on the molecular events that shape the outcome of Th1/2 differentiation.
| S-EPMC365738 | biostudies-literature