Hypoxia (low O2) plays a central role in a diverse array of human diseases. O2 is sensed by the hypoxia
response pathway comprising a prolyl hydroxylase (PHD) enzyme, which uses O2 to hydroxylate specific
prolines on the Hypoxia Inducible Factor a (HIFa). Once hydroxylated, HIFa is ubiquitinated by the Von
Hippel-Lindau (VHL) ubiquitin ligase, resulting in its proteolysis. When hypoxia ensues, PHD enzymes lack the
O2 to hydroxylate HIFa, resulting in HIFa stabilization, entry into the nucleus, and the transcriptional regulation
of multiple target genes. We currently do not know all of the proteins that regulate this pathway, how this
pathway is modulated in different tissue types, or how it uses a single O2 sensor with a low affinity for O2 to
respond to a broad dynamic range of O2 concentration. Because of the essential requirement of pathway
components in early development and viability in mammals, we also know little about how the pathway actually
works in vivo in an intact animal.
To address these questions, this proposal takes advantage of genetics and an intact, isogenic model
organism (C. elegans) that can thrive under hypoxia and whose environment and genetics can be controlled
with fidelity and reproducibility. C. elegans possess single genes for the PHD (EGL-9), the VHL (VHL-1), and
the HIFa (HIF-1). We recently identified four new regulators/mediators of this pathway. First, the PMK-1
ortholog of p38 MAPK promotes EGL-9 function under normoxia. Second, the EGL-4 ortholog of Protein
Kinase G (PKG) is a substrate of PMK-1 that is required for PMK-1 to regulate EGL-9 activity. Third, the PDR-
1 ortholog of the ubiquitin ligase Parkin inhibits HIF-1 in neurons. Fourth, the CHN-1 ortholog of the ubiquitin
ligase and chaperone CHIP, a factor known to work with Parkin, inhibits HIF-1 in neurons.
We hypothesize that PMK-1 regulates the pathway by activating EGL-4 via phosphorylation. We believe
that they allow for an additional layer of regulation, expanding the dynamic range of O2 sensation and
modulating the timing of the response. We also hypothesize that PDR-1 and CHN-1 form an ubiquitin ligase
pair that regulates HIF-1 independently of (and in different tissues from) regulation by VHL-1, thereby allowing
context-specific and tissue-specific patterns of hypoxia response. Here we will characterize the mechanism by
which CHN-1, PDR-1, EGL-4, and PMK-1 regulate the pathway in vivo. We will measure HIF-1 ubiquitination
and turnover, target gene expression, EGL-9 activity and subcellular localization, hypoxia survival, O2
consumption and ATP generation, oxidative stress, and mitochondrial dynamics in mutants for these factors.
We will directly test whether PDR-1 and CHN-1 regulate the pathway through HIF-1. We will test whether
PMK-1 regulates the pathway by phosphorylating EGL-4. We will use a proteomics approach to identify
downstream substrates of EGL-4 that operate as part of the pathway. At its conclusion, these studies will have
provided the foundation for examining whether the orthologs of these factors conduct similar roles in mammals.