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PMC:1208879 / 17654-24825
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1208879","sourcedb":"PMC","sourceid":"1208879","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1208879","text":"Discussion\nOn the basis of secondary structure predictions and amino acid sequence conservation, we defined a novel PAX6 protein domain, which we have called the C-terminal peptide. We performed yeast two-hybrid library screens with the C-terminal peptide and the whole PST domain and we identified three novel interacting proteins, HOMER3, DNCL1 and TRIM11. In library screens, HOMER3 and DNCL1 interacted with the C-terminal peptide and the PST domain while TRIM11 interacted only with the PST domain, suggesting that HOMER3 and DNCL1 specifically interact with the C-terminal peptide while TRIM11 interacts with a more N-terminal part of the PST domain. The interactions between the PST domain and HOMER3, DNCL1 and TRIM11 were confirmed in pairwise tests. We were not able to confirm the interaction between HOMER3 or DNCL1 with the C-terminal peptide construct in pairwise tests, but we showed that the C-terminal peptide was important for PAX6/HOMER3 or PAX6/DNCL1 interaction because HOMER3 and DNCL1 did not interact with a PST domain construct lacking the C-terminal peptide.\nHOMER3 is found in the PSD of neurons and directly binds to type I metabotropic glutamate receptors, which act via phospholipase C to stimulate IP3-mediated release of Ca2+ from intracellular vesicles [35,39]. HOMER3 is a member of the HOMER family of proteins that are constitutively expressed in the brain and play a role in post-synaptic signalling and receptor trafficking by forming multivalent links with various receptors and PSD scaffolding proteins [35,39-41]. HOMER proteins have also been implicated in axon guidance during brain development [42].\nDNCL1 is a subunit of two intracellular transport protein complexes, dynein and myosin Va [36]. Dynein and myosin Va are involved in the microtubule-based and actin-based movement respectively of proteins, organelles and vesicles in neurons [36,37]. Myosin Va is enriched in the PSD [43], and DNCL1 binds to a variety of PSD proteins including guanylate kinase domain-associated protein [44] and neuronal nitric oxide synthase [45].\nTRIM11 is a member of the mouse tripartite motif protein family (also known as the RBCC family), and contains the three characteristic structural motifs of this protein family, a RING finger, a B-box zinc finger, and a coiled coil domain, as well as a B30.2 domain that is found in many but not all TRIM proteins [38]. TRIM11 interacts with Humanin, a protein that suppresses the neurotoxicity associated with Alzheimer's disease [46]. TRIM11 lowers Humanin levels by a mechanism that appears to involve ubiqutin-mediated proteasomal degradation [46].\nAt present our data must be considered preliminary because the interactions have not been confirmed by any other approach. However it is interesting to speculate that the interaction of PAX6 with HOMER3 and DNCL1 may be the basis of a mechanism by which synaptic signalling causes changes in gene expression. We propose that PAX6 is sequestered in the PSD by binding to HOMER3. Since receptor activation causes dissociation of HOMER proteins [35], PAX6 may be released as a result of synaptic activity, allowing it to interact with DNCL1. The PAX6/DNCL1 complex could then participate in myosin Va-mediated transport along the PSD-associated actin cytoskeleton followed by dynein-mediated transport along the microtubule network, eventually reaching the nucleus [36]. Since TRIM11 is implicated in protein degradation [46], it may play a role in PAX6 protein turnover.\nPrecedents for association of transcription factors with the post-synaptic density include STAT3 and CREB, which act as messengers between the synapse and the nucleus [47,48]. Although the full-length PAX6 protein is predominantly nuclear, there is good evidence in mouse, quail and nematode for an isoform that lacks the paired domain, is both nuclear and cytoplasmic, and binds DNA through the homeodomain alone [49-51]. In mice the paired-less isoform is relatively abundant in brain [49]; however its subcellular localisation in neurones, and the possibility of an association with the PSD, remains to be investigated.\nRegarding the subcellular localisation of the putative interacting proteins, HOMER3 is predominantly found at the interface between the PSD and the cytoplasm [39], DNCL1 is chiefly cytoplasmic, although nuclear localisation has been reported [51], and TRIM11 is both nuclear and cytoplasmic [38]. Therefore cytoplasmic PAX6 could potentially interact with all three proteins, while nuclear PAX6 could interact with TRIM11 and DNCL1. At the tissue level, HOMER3 expression has been detected in thymus and lung but it has mainly been studied in brain where it is found in the forebrain, hippocampus and cerebellum [39]. DNCL1 and TRIM11 both have wide expression domains that include the brain [44,38]. Thus the expression of all three interactors overlaps with PAX6 at the tissue level [2,7,8,10]. We detected co-expression of PAX6, HOMER3, DNCL1 and TRIM11 by RT-PCR in human adult brain RNA (IH, L Harrison and A Brown, data not shown).\nWe demonstrated that the interaction between the PST domain and HOMER3 or DNCL1 was impaired by three naturally occurring mutations that are located in the PAX6 C-terminal peptide. The Q422R mutation, which involves a glutamine to arginine substitution at the last amino acid position of PAX6, caused a reduction in the interaction with HOMER3 and DNCL1. The X423L and 1615del10 mutations severely reduced or completely abolished the interaction with HOMER3 and DNCL1. The predicted effect of the X423L and 1615del10 mutations is to cause translation into the 3' untranslated region, thus generating proteins with abnormal extensions that might be expected to disrupt the conformation of the C-terminal end of the protein. The 1615del10 mutation also removes the last 5 amino acids of the C-terminal peptide [28].\nNone of the mutations affected the interaction with TRIM11, suggesting that they do not alter the conformation of the more N-terminal part of the PST domain. All our data are consistent with the hypothesis that TRIM11 does not interact with the C-terminal peptide, but interacts with the PST domain between the homeodomain and the C-terminal peptide.\nMost aniridia patients are heterozygous for mutations that introduce a premature termination codon into the PAX6 open reading frame [2,52]. These alleles would be expected to encode truncated proteins or no protein at all if the mutant RNA is degraded by nonsense-mediated decay [52]. We propose that the brain anomalies that have been observed in aniridia patients may be partly explained by impaired interaction between PAX6 and HOMER3, DNCL1 and TRIM11. The neurobehavioural phenotype associated with 1615del10 and the polymicrogyria associated with X423L may result from a specific effect of these unusual C-terminal extension mutations. There is evidence that signalling and transport mechanisms that were initially characterized in the brain may also be conserved in the retina, suggesting that impaired PAX6 protein-protein interactions may also have implications for the retinal defects observed in individuals with PAX6 mutations 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