Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.
The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.
Disulfide linkages have long been known to stabilize the structure of many proteins. In recent work, this bond has also been classified as a reversible post-translational modification, acting as a cysteine-based "redox switch" allowing for the modulation of protein function, location and interaction1,2,3,4. Thus, it is important to be able to study this modification. A simple method to analyze these cysteine-stabilized multimeric complexes is through non-reducing SDS-PAGE analysis5. SDS-PAGE analysis is a technique used in many laboratories, where the results can be obtained and interpreted quickly, easily, and with minimal costs, and is advantageous over other techniques used to identify disulfide linkages such as mass spectrometry6,7 and circular dichroism8.
One important step in determining if this method is an appropriate technique to aid in a study is to thoroughly examine the primary sequence of the protein of interest to insure there are cysteine residue(s) present. Another helpful step is to research any previous crystal structures published or use a bioinformatics application to explore the three dimensional structure of the protein of interest to visualize where the cysteine residue(s) may be located. If the residue(s) is present on the outside surface it may be a better candidate to form a disulfide linkage rather than a cysteine residue buried on the inside of the structure. However, it is important to note that proteins may undergo structural changes upon substrate interactions or protein-protein interactions allowing these residues to then become exposed to the environment as well.
Identified multimeric complexes can then be verified with chemical cross-linking using formaldehyde. Formaldehyde is an ideal cross-linker for this verification technique due to the high cell permeability and short cross-linking span of ~2-3 Å, ensuring detection of specific protein-protein interactions9,10. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS11. However, this protocol can be adapted for other cell lines, tissues and organisms.
1. Blocking Free Cysteine Residues Using Iodoacetamide
2. Harvesting the Cells
3. Extraction of Protein
4. Sample Preparation
5. In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells
6. Fractionation of Nuclei
7. Extraction of Protein
8. Sample Preparation
9. SDS-PAGE Analysis
10. Western Blot
Nuclear dUTPase forms an intermolecular disulfide linkage forming a stable dimer configuration through the interaction of two cysteine residues positioned at the third amino acid of each monomeric protein11. This is demonstrated in Figure 1A,B. To ensure this disulfide linkage was not a nonspecific interaction due to migration abnormalities in the non-reduced environment,t the inclusion of a proper control was essential. Of note, nuclear dUTPase is one of four isoforms present in humans. Three of the four isoforms have a unique amino-terminal domain while sharing a common catalytic core11,12. As seen on the western blot, the monomeric confirmation of dUTPase in human cells at the time of harvesting is a combination of at least three of the isoforms of dUTPase (the mitochondria isoform, the nuclear isoform, and a truncated version notated at M24), all of which are recognized by our polyclonal antibody. The nuclear isoform is the only isoform that contains a cysteine residue in its unique amino terminal domain. Due to the nuclear isoform being only a small percentage of the monomeric state of the proteins seen on the western blot, it was exposed longer to demonstrate the dimeric state of that isoform.
The mitochondrial isoform lacks the cysteine residue present in nuclear isoform and was used as a control for this intermolecular disulfide linkage. As seen in Figure 1C, isolation of mitochondria followed by a western blot analysis demonstrated that this isoform under non-reducing conditions did not form a disulfide linkage and migrated to the predicted molecular weight for the monomeric protein.
To confirm this complex can form a multimeric complex, formaldehyde cross-linking was performed. Isolated nuclei were subjected to 1% formaldehyde treatment followed by denaturing SDS-PAGE/western blot analysis under reducing conditions. As demonstrated in Figure 2, the dimerization was visualized. When the cross-link was reversed by incubating the sample at 95 °C for 15 min, the complex was destabilized and could be visualized in its monomeric state.
Figure 1: Demonstration of intermolecular disulfide bond formation in the nuclear dUTPase protein. (A) A western blot analysis of total cell extracts (TCE) in the absence of the reducing agent, beta-mercaptoethanol (BME), demonstrates a multimeric complex formation in asynchronous populations of U-2 OS, Saos2, A549, and 18CO as indicated by the black box. (B) This complex disappears with the addition of BME in all four cell lines examined, indicating the presence of a disulfide linkage. (C) A western blot analysis of TCE and purified mitochondrial extracts (Mito) derived from U-2 OS cells, ±BME, shows no multimeric complex formation in the -BME sample. The lower panels in panels A, B, and C demonstrate exposure to X-ray film for 10 s, showing the monomeric state of the three isoforms of dUTPase. The upper panels in A and B were exposed for 1 min, while the upper panel in C was exposed for 2 min. Equivalent amounts of protein were applied to each lane. Blots were probed with a polyclonal specific antibody against the conserved carboxyl-terminal domain of dUTPase. This figure has been modified from Rotoli et al.11 Please click here to view a larger version of this figure.
Figure 2: Formaldehyde cross-linking of nuclear dUTPase demonstrate multimeric complex formation. U-2 OS cells were incubated with 1% formaldehyde for 15 min. Nuclei (N) were isolated then analyzed by western blot using a specific polyclonal antibody against the conserved carboxyl-terminal domain of dUTPase (+ formaldehyde). To reverse the formaldehyde cross-links, extracts derived from the nuclear preparations were mixed with SDS-PAGE buffer then heated to 98 °C for 15 min in the presence of BME (+formaldehyde, 98 °C for 15 min). The observed heterogeneity (i.e. doublet bands of nDut) seen with the preparations remain to be explained, but may be due to anomalous migration due to the formaldehyde treatment. The lower panel is exposed to X-ray film for 10 s, while the upper panel is exposed to X-ray film for 2 min. This figure has been modified from Rotoli et al.11 Please click here to view a larger version of this figure.
The method outlined here gives a straight-forward protocol for the analysis of multimeric complexes stabilized through disulfide linkages. This protocol can easily be adapted to other cell culture lines, tissues and organisms allowing for a broad range of applications.
An important step in this procedure is to ensure the disulfide linkages are not a consequence of the extraction procedure. Any free cysteine residues can be blocked using iodoacetamide13. This alkylating agent will bind covalently to cysteine residues though their thiol group, blocking the formation of new disulfide bonds. However, if the disulfide bond is present at the time of treatment this agent will not disrupt it. Optimization of the iodoacetamide can be done using a variation of concentration and time. However, over exposure to this reagent will cause cell death.
An additional step to this protocol that may be helpful is the optimization of the formaldehyde cross-linking. A variation of the percent of formaldehyde can be used as well as the time of cross-linking9,14. It is important to note that as the percent of formaldehyde as well as time increases, so do the chances of forming non-specific interactions. Downstream confirmation of the multimeric complex is also necessary. A useful technique to determine the molecular weight and identity, if the complex is a heteromultimeric, is mass spectrometry. Also, site directed mutagenesis can be a suitable technique to determine the cysteine residues responsible for the disulfide linkage.
Lastly, this two-step method of non-reduced SDS-PAGE analysis and formaldehyde cross-linking verification is beneficial due to its technical ease and low cost. It can be a first step in uncovering multimeric complexes stabilized by disulfide linkages.
The authors have nothing to disclose.
We gratefully appreciate the efforts put forth by Dr. Jennifer Fischer for the purification of the dUTPase polyclonal antibody and Kerri Ciccaglione for all her efforts to help edit this manuscript. This research was partially supported by a grant from the New Jersey Health Foundation (Grant #PC 11-18).
16% precast TGX gels | ThermoFisher | Xp00160 | |
175 cm2 Flask | Cell star | 658175 | |
18CO | ATCC | CRL-1459 | |
6 cm2 dish | VWR | 10861-588 | |
A549 | ATCC | CCL-185 | |
Amersham ECL detection kit | GE | 16817200 | |
Blot transfer apparatus | Biorad | 153BR76789 | |
BME | Sigma Aldrich | M3148 | |
Bradford protein reagent | Biorad | 5000006 | |
Bromophenol Blue | |||
BSA | Cell signaling | 99985 | |
Cell lysis buffer | Cell signaling | 9803 | |
Centrifuge | Eppendor | 5415D | |
DMEM | Gibco | 11330-032 | |
Drill | |||
EDTA | Sigma Aldrich | M101 | |
Electrophoresis apparatus | Invitrogen | A25977 | |
Extra thick western blotting paper | ThermoFisher | 88610 | |
Fetal bovine serum | Gibco | 1932693 | |
Formaldehyde | ThermoFisher | 28908 | |
Glass-teflon homogenizer | |||
Glycerol | Sigma Aldrich | 65516 | |
Glycine | RPI | 636050 | |
Heat block | Denville | 10285-D | |
Hepes | Sigma Aldrich | H0527 | |
Hydrochloric acid | VWR | 2018010431 | |
Iodoacetamide | ThermoFisher | 90034 | |
Kimwipe | Kimtech | 34155 | |
Methanol | Pharmco | 339000000 | |
Non-fat dry milk | Cell signaling | 99995 | |
PBS | Sigma Aldrich | P3813 | |
PMSF | Sigma Aldrich | 329-98-6 | |
Posi-click tube | Denville | C2170 | |
Power supply | Biorad | 200120 | |
Prestained marker | ThermoFisher | 26619 | |
PVDF membrane | Biorad | 162-0177 | |
Rocker | Reliable Scientific | 55 | |
Saos2 | ATCC | HTB-85 | |
SDS | Biorad | 161-0302 | |
Secondary antibody | Cell signaling | 70748 | |
Small cell scraper | Tygon | S-50HL class VI | |
Sodium chloride | RPI | S23020 | |
Sodium pyruvate | Gibco | ||
Sonicator | Branson | 450 | |
Sponge pad for blotting | Invitrogen | E19051 | |
Stir plate | Corning | PC353 | |
Sucrose | Sigma Aldrich | S-1888 | |
Tris Base | RPI | T60040 | |
Tris Buffered Saline, with Tween 20, pH 7.5 | Sigma Aldrich | SRE0031 | |
Tris-Glycine running buffer | VWR | J61006 | |
Triton X-100 | Sigma Aldrich | T8787 | |
Tween 20 | Sigma Aldrich | P9416 | |
U-2 OS | ATCC | HTB-96 | |
X-ray film | ThermoFisher | 34090 |