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Enzymes02:34

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Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
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Esterification Delivers a Functional Enzyme into a Human Cell.

Valerie T Ressler1, Kalie A Mix1, Ronald T Raines1

  • 1Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States.

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|March 5, 2019
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Researchers developed a method to deliver active proteins into mammalian cell cytosol. Esterifying enzyme carboxyl groups with a diazo compound allows protein entry and retains catalytic function, offering new possibilities for cellular therapies.

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Area of Science:

  • Chemical biology
  • Cellular biology
  • Biochemistry

Background:

  • Delivering native proteins into the cytosol of mammalian cells presents a significant challenge in chemical biology.
  • Existing methods often struggle with protein activity loss or inefficient cellular uptake.

Purpose of the Study:

  • To develop a novel strategy for the effective delivery of functional enzymes into the cytosol of mammalian cells.
  • To demonstrate the retention of catalytic activity after intracellular protein delivery.

Main Methods:

  • Esterification of enzyme carboxyl groups using a diazo compound.
  • Cellular uptake studies of modified enzymes in mammalian cells.
  • Assessment of enzyme activity post-internalization and hydrolysis of ester bonds.

Main Results:

  • The esterification strategy successfully facilitated the passage of enzymes into the cell cytosol.
  • The delivered enzymes retained their native catalytic activity within the cellular environment.
  • Internalized esters were hydrolyzed by endogenous esterases, resulting in a traceless delivery process.
  • Human ribonuclease 1 demonstrated retained cytotoxic ribonucleolytic activity after delivery.

Conclusions:

  • Enzyme esterification with diazo compounds is an effective method for traceless protein delivery into the cytosol.
  • This approach preserves enzyme catalytic function, opening new avenues for therapeutic enzyme applications.
  • The strategy holds significant promise for advancing chemical biology and cell-based therapies.