NO signaling by a Soluble Guanylyl Cyclase -Thioredoxin transnitrosation complex

PROJECT SUMMARY
Nitric oxide (NO) is an important signaling molecule that regulates diverse functions relevant to vascular
function, apoptosis and angiogenesis. NO is best known for its ability to stimulate soluble guanylyl
cyclase (now called GC1) to produce cGMP and stimulate its downstream signaling pathways.
However, NO can also covalently modify cysteines (Cys) via S-nitrosation or S-nitrosylation (addition
of a NO moiety to the cysteine of a protein, SNO). Although this reversible post-translational
modification is increasingly recognized as an important regulatory mechanism of protein function,
dynamic regulation of protein nitrosation specificity is poorly understood. Our most recent investigations
reveal that GC1 has a transnitrosylase activity, i.e. GC1 has the ability to directly transfer SNO to
specific targets by protein-protein interaction (transnitrosation). This transnitrosation activity does not
require the cGMP forming activity of GC1 and can be accomplished by a single subunit of GC1
(formation of cGMP requires 2 subunits). Furthermore, we showed that one transnitrosation target of
GC1 is oxidized thioredoxin 1 (oTrx1), a thiol-redox protein that modulates cellular S-nitrosation. In fact,
oxidative/nitrosative conditions appear to favor the GC1-Trx1 complex. Using advanced proteomics
approaches, we recently identified the Cys in GC1 and Trx1 that are involved in the SNO transfer in a
purified system, and the Cys of proteins targeted by the GC1/Trx1 transnitrosation cascade in smooth
muscle and cardiac cells. Our hypothesis is that the function of GC1 transnitrosation activity is an
adaptive response to oxidative stress and potentially compensates for the dysfunction of the canonical
NO-GC1-cGMP pathway that occurs in oxidative conditions. To explore this provocative hypothesis,
we propose to conduct mutational analysis of the Cys we have identified to characterize the mechanism
of transnitrosation in smooth muscle and cardiac cells. By comparing the targets of GC1, Trx1 and both
we will determine the mechanisms underlying target specificity. We will determine how GC1/Trx1
transnitrosation of specific targets affects their cellular function. For this, we will use cell lines and
primary cells isolated from a novel mouse knock-in (KI) of a Cys of GC1 involved in transnitrosation.
To determine the physiological relevance of GC1- and GC1/Trx1-transnitrosation in the cardiovascular
system and the adaptive response to stress, we will use the Cys KI mouse model and inhibitory peptides
that disrupt the GC1/Trx1 transnitrosating complex under Angiotensin II-induced oxidative stress. This
project could lead to the discovery of novel cardiovascular protective pathways driven by specific S-
nitrosation.

Grant Number: 5R01GM112415-08
NIH Institute/Center: NIH
Principal Investigator: ANNIE BEUVE