Structural and functional characterization of IMPACT proteins: a novel nuclease family

IMPACT proteins are known to be present in almost all cell types, but they are highly expressed in fibroblasts and central nervous system. The term IMPACT comes from the fact that the Impact gene in rodents is the only imprinted gene found at chromosome 18 up to date. This imprinting is not found in other mammalian species. The protein is divided in two domains, an RWD domain at the N-terminus and the ancient domain at the C-terminus. Between those domains there is a linker region with no predicted structure. All IMPACT proteins display very high sequence conservation, mostly on its ancient domain. However, there is no known function of the protein related to the ancient domain. Little is known about the role of IMPACT proteins within the cell. Nowadays, the only well-known function of IMPACT proteins is to assure translation levels upon amino acid starvation conditions. Protein translation is a crucial process for life. Hence, adaptation to stress conditions that compromise translation is a matter of survival. IMPACT proteins are able to compete with GCN2 for the binding to GCN1, since both GCN2 and IMPACT harbour an RWD domain able to interact with GCN1. GCN2 binding to GCN1 is necessary to activate the kinase domain of GCN2. The activation of the kinase domain results in eIF2 phosphorylation at serine 51 on its alpha subunit. Phosphorylation of eIF2α leads to general translation repression but it activates the expression of some transcription factors involved into cell remediation and amino acid biosynthetic pathways. Since it is overexpressed in central nervous systems it may be involved in the regulation of translation in some specific neuronal cells upon amino acid starvation. On the other hand, it has been described that IDO induction is able to provoke IMPACT overexpression by tryptophan depletion. IDO activity is based on tryptophan catabolism into toxic compounds known as kinenurines. Regarding its role in translation regulation, IMPACT proteins would help cells to overcome tryptophan depletion. Recent studies are trying to shed light into new possible roles of IMPACT in the cell biology have linked IMPACT proteins with cell cycle, since it has been demonstrated that IMPACT and its yeast counterpart Yih1 are able to interact with CDK1 or cdc28 respectively, although the particular function of the interaction remains unknown. In this work we describe for the first time the three dimensional structure of the IMPACT RWD and ancient domains from a thermophilic fungus known as Chaetomium thermophilum. We have proved that IMPACT proteins are able to bind both, RNA and DNA in vitro. Extensive biochemical analysis from human, S.cerevisiae and C.thermophilum and detailed structural analysis of the obtained structures lead us to the identification of IMPACT proteins as enzymes that are able to endolytically cleave DNA. Besides, further characterization of the reaction revealed that the cleavage was not simultaneous in both strands and that the enzyme displays substrate inhibition. Given the cytosolic location of the protein, we wonder if IMPACT proteins would be related to immune system. In vitro experiments also demonstrated that IMPACT proteins act as cytosolic DNA sensors, since IMPACT knock down in mouse fibroblasts affects to interferon beta mRNA levels after transfection with ISD. In brief, these findings open a new field for the study of the roles of IMPACT proteins within cell biology.