The hard path towards accurately measuring in vivo enzyme activity: the case of protein disulfide isomerase

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by Denise C. Fernandes

Correct protein folding is a vital and extremely regulated cellular function. Disulfide bonds are essential determinants of the correctly folded protein structure. During the folding of nascent proteins into the endoplasmic reticulum (ER) lumen, essential enzymes promote disulfide bond insertion (oxidation) and their eventual repositioning (isomerization) when they are initially formed between wrong cysteines. These reactions are catalyzed by PDIs (protein disulfide isomerases), a family of enzymes that contains more than 20 members, from yeast to humans [1]. Thus, PDIs do not have one specific substrate, but rather a large variety of un/misfolded protein substrates.

Using purified PDIA1 (the founder member of PDI family), it is possible to measure 3 PDI redox activities: disulfide reduction, oxidation or isomerization, depending on the substrate selected for the assay. In cells, although most of PDIA1 resides in the ER, a small quantity is able to bypass ER retention mechanisms by so far unknown mechanisms, and is found in cytosol, nuclei, mitochondria and plasma membrane. PDIA1 function(s) in these subcellular compartments is not so clear, but in plasma membrane there are good evidences that PDIA1 is preferentially a reductase, acting in glycoproteins such as integrins – with implications in virus infection, thrombus formation and coagulation [2]. Thus, the preferential in vivo PDIA1 redox activity depends on compartmentalized redox environment. While in the more oxidizing ER lumen PDIA1 is rather an oxidase/isomerase, in the more reducing surrounding of plasma membrane PDIA1 behaves mostly as a reductase. Besides the strictly redox activities, PDIA1 exhibits a chaperone activity (i.e., assisting other proteins to stabilize), not directly dependent on its redox thiols. In vitro,it is also possible to measure solely PDIA1 chaperone activity, if the substrate chosen is devoid of disulfide bonds.

Putting all together, while with purified protein is possible to measure up to 4 activities (chaperone, thiol reduction, oxidation and isomerization), in vivo all these activities should overlap into each other to achieve PDIA1 function in each specific cellular compartment. These issues were recently critically discussed in a minireview about methods for measuring PDI activities in vitro and in cells [3]. Not surprisingly, there are interferents when measuring PDI activity in cellular samples. These include endogenous interferents such as other reductase enzymes, e.g. thioredoxin itself. Intriguingly, we described that several surfactants used in common buffers in procedures of lysis or preparation for microscopy analysis may alter PDI assays by inhibiting its reductase activity.

These methodological hardships are not unusual in essentially every research field and the task of choosing a specific method requires a lot of scrutiny, great involvement with the project and design of several controls to avoid intrinsic interferents. Particularly, in redox research, in which protein/cellular sample manipulation generates artifactual oxidations (such as disulfide formation) due to molecular oxygen, this is a more challenging task, especially when the fascinating multifaceted PDI is involved.

  1. F. R. Laurindo, L. A. Pescatore, D. C. Fernandes.
    Protein disulfide isomerase in redox cell signaling and homeostasis.
    Free Radical Biology & Medicine, 52 (9): 1954-69, 2012. |
  2. H. Ali Khan, B. Mutus.
    Protein disulfide isomerase a multifunctional protein with multiple physiological roles.
    Frontiers in Chemistry, 2: 70, 2014. |
  3. M. M. Watanabe, F. R. Laurindo, D. C. Fernandes.
    Methods of measuring protein disulfide isomerase activity: a critical overview.
    Frontiers in Chemistry, 2: 73, 2014. |

Denise de Castro Fernandes, Ph.D.
Associate Researcher of Vascular Biology Laboratory, Heart Institute (InCor),
University of São Paulo School of Medicine, São Paulo, Brazil

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