FCiências.ID Amphitheatre – April 16, 17h

Tomás Silva

PhD Student – CBS

Computational study of pH-dependent membrane insertion of pHLIP peptides

The  pH  (low)  insertion  peptide  (pHLIP)  belongs  to  a  family  of  transmembrane peptides, with the ability to insert in membrane cells dependent on how acidic is their vicinity, such as in tumoral cells, thus working as an efficient tumor-specific biomarker.  However,  wt-pHLIP  pH  -dependent membrane  insertion  has  not  been fully explored at the molecular  level. Also,  since the  peptide accumulates  in the kidneys in considerable amounts, due to their naturally acidic extracellular pH, it is urgent to improve peptide specificity by delimiting the pH range of insertion.

The  constant-pH  molecular  dynamics  (CpHMD)  method  proved  to  be  capable  in sampling protonation states of titrable residues of both wt-pHLIP and L16H variant, while  also  revealing  important  structural  details  about  the  peptide-membrane equilibria.  Furthermore,  our  work  produced  remarkable  results  by  predicting detailed and accurate pKa  profiles of the residues. Moreover, the L16H variant was able to establish a pH range of insertion, despite being too large for in vivo studies and therapeutic purposes. For the future, we aim to improve sampling quality, using a pH replica exchange (pHRE) methodology, for better system description and pKa prediction. Additionally, we will design new pHLIP sequences mutated with cationic residues, as to narrow down the pH range of insertion and evaluate their validity for clinical applications.

Márcia Faria

BioSYS PhD Student – FunGP

Targeting RAC1-signaling to enhance iodide-related cancer therapy

The  Sodium  Iodide  Symporter  (NIS),  responsible  for  active  transport  of  iodide  into thyroid  cells,  allows the  use  of  radioactive  iodine  (RAI)  as  the  systemic treatment  of choice for thyroid cancer (TC) metastatic disease. Still, patients with advanced forms of TC sometimes lose the ability to respond to RAI therapy, which reduces their survival rates. NIS protein abundance and stabilization at the plasma membrane (PM) has been put  forward  as  a  major  limiting  factor  for  iodide  uptake.  Thus,  in  this  study,  we  are currently investigating the impact of RAC1-signaling on NIS functional expression at the PM. We generated a TPC1 thyroid cancer cell line clone stably expressing a full-length NIS  construct  containing  an  extracellular  triple  HA  tag.  The  PM  levels  of  NIS  upon interference  with  RAC1-signaling  were  assessed  using  surface  protein  biotinylation assays.  Notably,  treatment  with  the  selective  RAC1  inhibitor,  EHT1864,  induced  a dramatic decrease (≈90%) on NIS overall and PM expression, while the overexpression of constitutively active RAC1 mutant produced the opposite result, increasing by over 2- fold NIS PM levels. To ascertain the potential impact of RAC1-signaling on the efficacy of iodide uptake, we used a non-radioactive iodide influx assay that showed a decreased iodide  influx  rate  upon  EHT1864  treatment.  These  results  disclose  an  unequivocal association between RAC1-signaling and NIS posttranslational regulation.