Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. To gain deeper insight into this pathway and its influence on cancer risk, we searched for novel components through protein physical interaction screens.
N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca²(+)-permeable cation channels which are blocked by extracellular Mg²(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg²(+) block and a decrease in Ca²(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.
Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-alpha subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin alpha/beta dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of "classical" importin alpha/beta receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.
Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-? subunits and a HIF-? subunit. Under normoxic conditions the HIF-? subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1? stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1? occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1? and shows less prolyl hydroxylase activity for its target HIF-1? than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1? predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.
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