Related Experiment Videos
Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper
1Molecular Biology Programme, National Public Health Institute, Helsinki, Finland.
Abstract:
Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.
Insights
Mouse Mx1 protein, an antiviral factor, forms dimers and trimers. A leucine zipper domain at its C-terminus drives oligomerization and nuclear localization, influencing its structure and function.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Interferons induce proteins with antiproliferative, antiviral, and immunomodulatory functions.
- Mx proteins are key mediators of the antiviral response, inhibiting viruses like influenza.
- Mouse Mx1 protein's structure and characteristics were largely unknown, despite its antiviral role.
Purpose of the Study:
- To biochemically characterize the mouse Mx1 protein.
- To investigate the structural basis of Mx protein oligomerization and nuclear localization.
- To elucidate the role of the C-terminal leucine zipper domain in Mx protein function.
Main Methods:
- Expression and purification of mouse Mx1 protein using a baculovirus system.
- Biochemical analysis of protein oligomerization states (dimers, trimers, aggregates).
- Cross-linking experiments, in vitro translation of chimeric constructs, and microinjection studies.
- Immunoelectron microscopy to determine the subcellular localization of nuclear murine Mx1 protein.
Main Results:
- Purified mouse Mx1 protein exists as dimers and trimers in solution and forms aggregates in physiological buffers.
- Mouse, rat, and human Mx proteins predominantly form trimers, mediated by a C-terminal leucine zipper domain.
- The C-terminus also acts as a nuclear localization signal, directing protein accumulation in the nucleus.
- Nuclear murine Mx1 protein localizes to distinct electron-dense structures.
Conclusions:
- The C-terminal leucine zipper domain is a critical structural element for Mx protein oligomerization and nuclear localization across species.
- Mx protein oligomerization and nuclear localization are important for its biological functions, though the exact relevance is yet to be determined.