No

Fig. 2. NAP interacts with NOS2 monomers and prevents NOS2 homodimerization and NO synthesis.

denaturing gel and then immunoblotted, using antibody to NOS2. NOS2 is normally found in cells as a monomer (approximate Mr 140,000) and as a homo-dimer (approximate Mx 280,000). However, coexpression of NAP-110 alters the relative mobility of NOS2: no NOS2 monomers are detected, and a novel NAP-NOS2 complex is detected—and the proportion of NOS2 homodimers are greatly decreased.

These experiments demonstrate that NAP-110 interacts with NOS2: NAP-110 inhibits NOS2 by inhibiting formation of the active NOS2 homodimer (Fig. 2).

Conclusions

The use of the yeast two-hybrid system has facilitated the identification of polypeptides that potentially interact with the NOS isoforms. Because the yeast two-hybrid system can produce false positives, demonstration of an interaction in mammalian cells is necessary. The functional consequences of any protein interacting with NOS can be explored by assessing NOS localization, NOS activity, and NOS dimerization in the absence and presence of the interacting protein. Identification of the biological effects of NOS interactors may reveal novel physiological mechanisms of regulating NOS.

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