Methods to identify the substrates of thiol-disulfide oxidoreductases

Abstract

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active-site motif embedded in their thioredoxin-like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol-disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram-negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.

Keywords: disulfide bond; disulfide-linked enzyme-substrate complex; oxidative protein folding; protein disulfide isomerase; thiol-based redox regulation; thioredoxin superfamily member.

Figure 1

Mechanisms of thiol‐disulfide exchange reactions catalyzed by thiol‐disulfide oxidoreductases. Formation (A), isomerization (B), and reduction (C) of a disulfide bond.

Figure 2

Importance of pretreatment of cells with TCA for stabilizing disulfide‐linked complexes using NEM. (A and B) HeLa cells were grown in DMEM medium supplemented with 10% FBS for 48 h, and washed twice with PBS. Following this step, free cysteines in the sample were modified using two different protocols. In the first protocol, the cells were directly lysed and alkylated in TritonX‐100 buffer [50 mM Tris–HCl (pH 7.4), 1% (v/v) TritonX‐100, 150 mM NaCl, 2 mM EDTA] containing 20 mM NEM (Lanes 1 and 5) or 50 mM NEM (Lanes 3 and 7). In the second protocol, the cells were first treated with 10% TCA, washed twice with ice‐cold acetone, and dissolved in SDS buffer [100 mM Tris–HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS)] containing 20 mM NEM (Lanes 2 and 6) or 50 mM NEM (Lanes 4 and 8). Samples were separated by SDS‐polyacrylamide gel electrophoresis and analyzed by immunoblotting with either rabbit polyclonal antibody against PDI (Panel A) or rabbit polyclonal antibody against P5 (Panel B). Each lane contains 5 μg of proteins. Samples were reduced with 100 mM DTT before electrophoresis in Lanes 5–8. The positions of molecular mass standards are indicated on the left in kDa. The positions of the monomeric form of PDI and P5 are indicated on the right. Disulfide‐linked complexes involving either PDI or P5 are indicated on the right of Lanes 2 and 4 by vertical lines. A non‐specific band is indicated by asterisk on the right of the Panel B.

Figure 3

Stabilization of disulfide‐linked complexes in a tissue. See text for details.

Figure 4

Separation of the partners of an enzyme by non‐reducing‐reducing 2D gel electrophoresis.30, 31, 34, 48, 49 Disulfide‐linked complexes containing an enzyme of interest are purified from NEM‐alkylated cell or tissue lysate and separated under non‐reducing conditions. The gel lane is then excised from the gel, incubated in SDS sample buffer containing 5% β‐mercaptoethanol, and placed on the top of the second gel. After electrophoresis, proteins are visualized with an appropriate method such as silver staining. Gray band or spot, non‐specific proteins or non‐covalent interactors of the enzyme; red band, disulfide‐linked complexes between the enzyme and its partners; black band or spot, monomeric enzyme; faint blue dot, disulfide‐linked partners of the enzyme. A non‐specific protein or non‐covalent interactor of the enzyme will migrate on a diagonal line (A). The enzyme (E) contained in a disulfide‐linked complex (B) will migrate at the size of the enzyme (C) in the second dimension. The disulfide‐linked partner (D) of the enzyme in the complex will migrate on the off‐diagonal line, allowing the easy identification of the spots corresponding to the partners of the enzyme.

Figure 5

Identification of the partners of an endogenous enzyme after separation of disulfide‐linked complexes containing the enzyme by regular one‐dimensional non‐reducing gel electrophoresis. (A and B) Disulfide‐linked complexes containing an enzyme of interest are purified from NEM‐alkylated cell or tissue lysate using an antibody to the enzyme, separated by non‐reducing gel electrophoresis, and detected with an appropriate protein‐staining method such as silver staining (Panel A, Lane 2).32, 59 Lane 1 contains proteins purified from the same lysate using control IgG and serves as a negative control. Gray band, non‐specific proteins; black band, monomeric enzyme; red band, disulfide‐linked complexes between the enzyme and its partner proteins. (Panel B) Individual bands corresponding to the disulfide‐linked complexes (red band in Panel A) or a region of the gel containing the complexes (indicated by a black vertical line on the right of Lane 2 in Panel A) are excised from the gel, digested by trypsin, and analyzed by mass spectrometry. The obtained data is analyzed using an appropriate search program to identify the proteins contained in the sample. The same analysis can be performed with a sample obtained by immunopurification using control IgG to exclude non‐specific proteins from the list of the potential partners of the enzyme.59