AKTIP is a lamin-interacting factor essential for cell survival, implicated in telomeric metabolism and DNA replication. Four main observations link AKTIP to HGPS: i) AKTIP impairment recapitulates HGPS phenotype in vitro; ii) AKTIP impairment recapitulates HGPS phenotype in mice; iii) AKTIP partially co-localizes with lamins, and iv) AKTIP is mislocalized in patient-derived HGPS cells. In addition, we have observed a direct interaction of AKTIP with the replication factor PCNA. We postulate the hypothesis that an AKTIP complex enriched at the nuclear periphery including the replication factor PCNA, lamins and DNA acts as a checkpoint for challenging replicative events, e.g. replication fork stalling at telomeres. We expect that in HGPS cells this checkpoint is compromised, which, in turn, may contribute to the HGPS phenotype. We propose to extensively analyze AKTIP function in vitro, in patient-derived HGPS cells, and in LV-progerin infected human fibroblasts. We will use multiple AKTIP-tag fusions, advanced microscopy and ChIP-seq. To investigate the role of AKTIP in vivo, we will study mouse derived mesenchymal stem cells and produce stroma-targeted depletion of the mouse orthologue of AKTIP, Ft1. We expect that this research will give new insights into the connection between progerin and telomere dysfunction through AKTIP, along with information on the role of DNA replication impairment as a potential driver mechanism in progeria. Given that the knowledge of the determinants and driver mechanisms of HGPS etiology has not been yet fully acquired, we believe that studies on new lamin-interacting players, as AKTIP, will be instrumental to dissect the mechanistic bases of HGPS and to open the path to therapeutic strategies.
The best characterized form of progeria is the Hutchinson Gilford Progeria Syndrome (HGPS) for which the genetic basis has been defined: a mutation in exon 11 of the LMNA gene, which induces the expression of a cryptic splice site, in turn determining the production of a truncated form of lamin A, named progerin [1,2]. HGPS patients appear normal at birth, but develop severe multi-organ abnormalities within two years, including skeletal defects and absence of subcutaneous fat. Death occurs in the teenage years and is prevalently caused by heart attacks and strokes.
While the genetic cause of HGPS is defined, the pathways generating the disease phenotype are not yet fully clarified, both qualitatively and hierarchically. Here we propose to investigate the role in HGPS etiology of AKTIP, a protein that is essential for cell survival and involved in telomere replication.
The rationale for studying AKTIP in the context of HGPS comes from four main observations: i) AKTIP impairment recapitulates HGPS phenotype in vitro; ii) AKTIP impairment recapitulates HGPS phenotype in vivo; iii) AKTIP partially co-localizes with lamins, and iv) AKTIP is mislocalized in HGPS patient cells. Along with this, we have shown that AKTIP biochemically interacts with lamin A/C and with lamin B1, with the replication factors PCNA and RPA70, and with the telomeric proteins TRF1 and TRF2 [1, 2].
We hypothesize that in wild type cells, AKTIP functions in DNA (telomere) replication as part of dynamic protein complex involving lamins, PCNA and replicating DNA. However, in HGPS cells, AKTIP mislocalization causes complex disorganization, DNA replication dysfunction and DNA damage, which would contribute to the progeroid phenotype.