Modulación de la proteostasis celular por el inhibidor ala-ala-phe-clorometilcetona

Supervised by:
  1. Luis Antón Canto Director
  2. Margarita del Val Latorre Co-director

Defence university: Universidad Autónoma de Madrid

Fecha de defensa: 04 April 2013

  1. José Javier Lucas Lozano Chair
  2. José María Requena Solanía Secretary
  3. Manuel Salvador Rodriguez Medina Committee member
  4. Alicia Mansilla Aparicio Committee member
  5. Carlos Guillén Committee member

Type: Thesis

Teseo: 354817 DIALNET


Ala-Ala-Phe-chloromethylketone (AAF-cmk) is a serine protease inhibitor that inhibits tripeptidyl peptidase II (TPPII), an enzyme that has been suggested to take up some proteasome functions when its activity is compromised. Similarly to proteasome inhibitors, AAF-cmk induces the accumulation of polyubiquitylated proteins including the formation of aggresome-like structures. This response is cell-dependent but it is not due to the inhibition of TPPII. We have proposed a working model where AAF-cmk could be acting upstream the proteasome by increasing the workload of the ubiquitin-proteasome system (UPS), which may result in a saturation of the proteasome in cells with a compromised activity of this degradative pathway. One of the aforementioned candidates could be Hsp90. Using the geldanamycin analog 17-AAG we showed that although Hsp90 is not a target of AAF-cmk, its inhibition leads to similar effects as those obtained with AAF-cmk. Given that the deubiquitylating activity is also upregulated in some cells with low proteasomal activity, and that its inhibition may result in an increase in the workload of the UPS, we decided to test whether AAF-cmk was affecting the activity of this group of enzymes. We have identified some activities that are inhibited by AAF-cmk such as USP7, USP14 and partially USP10. Taking into account that AAF-cmk has selective toxicity for some tumor cell lines, we evaluated the effect of the inhibitor on proteotoxic stress. Our results showed differences in the induction of cellular stress in two oteosarcoma cell lines correlating with agressome formation. Furthermore, we have observed that AAF-cmk also induces alterations on the autophagy pathway, Atg4B being another of its targets. In relation with new therapeutic targets it is important to analyze the synergism between the new drug and others which are already available. We have analyzed the effect of AAF-cmk in combination with the PI3K inhibitor, LY294002. The results showed an induction of both cell cycle arrest and cell death after AAF-cmk treatment, an effect that was enhanced by the combination of both inhibitors.