Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

Signal amplification in biomolecular networks converts a linear input to a steeply sigmoid output and is central to a number of cellular functions including proliferation, differentiation, homeostasis, adaptation, and biological rhythms. One canonical signal amplifying motif is zero-order ultrasensitivity that is mediated through the posttranslational modification (PTM) cycle of signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant, as by the classical Koshland–Goldbeter model. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here, we use mathematical models to explore the signal amplification properties in such scenarios and report some novel aspects. Our analyses indicate that PTM-induced protein stabilization brings the enzymes closer to saturation. As a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response, higher magnitude, and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Importantly, ultrasensitivity is not limited to modified or unmodified protein substrates—when protein turnover is considered, the total free protein substrate can also exhibit ultrasensitivity under several conditions. When full enzymatic reactions are used instead of Michaelis–Menten kinetics for the modeling, the total free protein substrate can even exhibit nonmonotonic dose–response patterns. It is conceivable that cells use inducible protein stabilization as a strategy in the signaling network to boost signal amplification while saving energy by keeping the protein substrate levels low at basal conditions.

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Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

10.1101/2021.08.22.457287 ◽

2021 ◽

Author(s):

Carla Kumbale ◽

Eberhard Voit ◽

Qiang Zhang

Keyword(s):

Posttranslational Modification ◽

Signal Amplification ◽

Covalent Modification ◽

Zero Order ◽

Protein Stabilization ◽

Signaling Proteins ◽

Cellular Functions ◽

Protein Substrate ◽

Protein Substrates ◽

Inducible Protein

Signal amplification converts a linear input to a steeply sigmoid output and is central to cellular functions. One canonical signal amplifying motif is zero-order ultrasensitivity through the posttranslational modification (PTM) cycle signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here we use a mathematical model to explore the signal amplification properties in such scenarios. Our simulations indicate that PTM-induced protein stabilization brings the enzymes closer to saturation, and as a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response of higher magnitude and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Interestingly, ultrasensitivity is not limited to modified or unmodified protein substrates; the total protein substrate can also exhibit ultrasensitivity. It is conceivable that cells use inducible protein stabilization as a way to boost signal amplification while saving energy by keeping the protein substrate at low basal conditions.

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Faculty Opinions recommendation of A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022696.261641 ◽

2004 ◽

Author(s):

Blake R Peterson

Keyword(s):

Covalent Modification ◽

Living Systems

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Faculty Opinions recommendation of Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725580616.793522620 ◽

2016 ◽

Author(s):

Morten Meldal ◽

Sanne Schoffelen

Keyword(s):

Covalent Modification ◽

Bioactive Proteins ◽

Synthetic Hydrogels

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Faculty Opinions recommendation of RNF4-Dependent Oncogene Activation by Protein Stabilization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726764766.793530897 ◽

2017 ◽

Author(s):

Anja-Katrin Bielinsky

Keyword(s):

Protein Stabilization ◽

Oncogene Activation

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Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191202145351 ◽

2020 ◽

Vol 21 (8) ◽

pp. 741-747

Author(s):

Liguang Zhang ◽

Yanan Shen ◽

Wenjing Lu ◽

Lengqiu Guo ◽

Min Xiang ◽

...

Keyword(s):

Tensile Strength ◽

Protein Function ◽

Protein Stabilization ◽

Functional Protein ◽

Elongation At Break ◽

Gelatin Films ◽

The Stability ◽

And Function ◽

General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

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Protein Solvent Interaction: Transition of Protein-solvent Interaction Concept from Basic Research into Solvent Manipulation of Chromatography

Current Protein and Peptide Science ◽

10.2174/1389203718666171024121529 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34-39 ◽

Cited By ~ 1

Author(s):

Tsutomu Arakawa ◽

Yoshiko Kita

Keyword(s):

Column Chromatography ◽

Rapid Development ◽

Academic Research ◽

Basic Research ◽

Protein Stabilization ◽

Special Issue ◽

Solvent Interaction ◽

Protein Formulations ◽

Solvent Additive ◽

Downstream Processes

Previously, we have reviewed in this journal (Arakawa, T., Kita, Y., Curr. Protein Pept. Sci., 15, 608-620, 2014) the interaction of arginine with proteins and various applications of this solvent additive in the area of protein formulations and downstream processes. In this special issue, we expand the concept of protein-solvent interaction into the analysis of the effects of solvent additives on various column chromatography, including mixed-mode chromatography. Earlier in our research, we have studied the interactions of such a variety of solvent additives as sugars, salts, amino acids, polymers and organic solvents with a variety of proteins, which resulted in mechanistic understanding on their protein stabilization and precipitation effects, the latter known as Hofmeister series. While such a study was then a pure academic research, rapid development of genetic engineering technologies and resultant biotechnologies made it a valuable knowledge in fully utilizing solvent additives in manipulation of protein solution, including column chromatography.

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HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

Current Cancer Drug Targets ◽

10.2174/1568009617666170315162525 ◽

2018 ◽

Vol 18 (3) ◽

pp. 287-294 ◽

Cited By ~ 8

Author(s):

Gustavo Alencastro Veiga Cruzeiro ◽

Maristella Bergamo dos Reis ◽

Vanessa Silva Silveira ◽

Regia Caroline Peixoto Lira ◽

Carlos Gilberto Carlotti Jr ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Cellular Proliferation ◽

Mrna Level ◽

Relevant Information ◽

Protein Stabilization ◽

Mrna Levels ◽

Medulloblastoma Cell Line ◽

Pediatric Medulloblastoma ◽

Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

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