Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

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AUTOCATALYTIC ACTIVATION OF Cls

Proteolysis and Physiological Regulation ◽

10.1016/b978-0-12-588250-7.50032-4 ◽

1976 ◽

pp. 400

Author(s):

Paul H. Morgan ◽

Indira G. Nair

Keyword(s):

Autocatalytic Activation

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Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014478 ◽

2020 ◽

Vol 295 (37) ◽

pp. 13047-13064 ◽

Cited By ~ 1

Author(s):

Elfriede Dall ◽

Florian B. Zauner ◽

Wai Tuck Soh ◽

Fatih Demir ◽

Sven O. Dahms ◽

...

Keyword(s):

Crystal Structure ◽

Arabidopsis Thaliana ◽

Cyclic Peptides ◽

Molecular Mechanisms ◽

Functional Studies ◽

Plant Characteristic ◽

Activation Mechanisms ◽

Binding Pockets ◽

First Time ◽

Autocatalytic Activation

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

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A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome

Nature Communications ◽

10.1038/ncomms10900 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 51

Author(s):

Eva M. Huber ◽

Wolfgang Heinemeyer ◽

Xia Li ◽

Cassandra S. Arendt ◽

Mark Hochstrasser ◽

...

Keyword(s):

20S Proteasome ◽

Autocatalytic Activation

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FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

The Journal of General Physiology ◽

10.1085/jgp.21.5.601 ◽

1938 ◽

Vol 21 (5) ◽

pp. 601-620 ◽

Cited By ~ 43

Author(s):

M. Kunitz

Keyword(s):

Temperature Coefficient ◽

Acid Medium ◽

Experimental Error ◽

Protein Concentration ◽

Liquid Culture ◽

Culture Medium ◽

Specific Activity ◽

Crystalline Form ◽

Liquid Culture Medium ◽

Autocatalytic Activation

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q5–15 = 1.70 to Q25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q10 = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.

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SOME PROPERTIES OF THROMBIN PREPARATIONS

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y59-084 ◽

1959 ◽

Vol 37 (1) ◽

pp. 775-785 ◽

Cited By ~ 2

Author(s):

Walter H. Seegers ◽

Gerardo Casillas ◽

Robert S. Shepard ◽

William R. Thomas ◽

Paul Halick

Keyword(s):

Esterase Activity ◽

Sodium Citrate ◽

Citrate Solution ◽

Ideal Model ◽

Terminal Amino Acid ◽

Two Stage ◽

Analytical Reagents ◽

Terminal Amino ◽

Autocatalytic Activation ◽

Prothrombin Activation

Bio-resin thrombin preparations were found to contain three weak precipitinogens. The clotting activity was not demonstrably associated with the precipitinogenic systems. Further, work was done on methods for the purification of citrate resin thrombin, and its clotting activity is also not associated with a precipitinogenic system. The N-terminal amino acid of both bio-resin thrombin and citrate resin thrombin was found to be glutamic acid. The two preparations were found to be homogeneous upon ultracentrifugal examination and could not be differentiated on the basis of sedimentation constants. Since "citrate" activation and "bio" activation produce eventually similar thrombin material, the autocatalytic activation of prothrombin in 25% sodium citrate solution can be used as an ideal model of prothrombin activation. The prothrombin first dissociates to form a derivative that does not form thrombin in the two-stage analytical reagents. Then a second alteration occurs in which the derivative again may form thrombin in the two-stage analytical reagents. Then thrombin activity appears as esterase activity, then as clotting activity. Later the clotting activity may be lost and finally also the esterase activity. The original prothrombin is a precipitinogen while the active thrombin is not.

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