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Teodoro Duarte Garcia Morais, Fernando Jorge
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- A conformational-dependent interdomain redox relay at the core of protein disulfide isomerase activityPublication . Pinho Melo, Eduardo; El-Guendouz, Soukaina; Correia, Cátia; Teodoro Duarte Garcia Morais, Fernando Jorge; Lopes, CarlosProtein disulfide isomerases (PDIs) are a family of molecular chaperones resident in the endoplasmic reticulum (ER) emerging as important factors in disease. In addition to an holdase function, some members catalyse disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by a modular arrangement of thioredoxin-like domains, with the canonical, first identified PDIA1, organized as four thioredoxin-like domains forming a horseshoe with two active sites at the extremities. Using two fluorescent redox sensors, roGFP2 and HyPer, as client substrates either unfolded or native, and the in vitro reconstitution of the full pathways of oxidative protein in the ER, we clarified important aspects underlying the catalytic cycle of PDIA1. The N-terminal a active site is the main oxidant of thiols and can transfer electrons to the C-terminal a´ active site relying on the redox-dependent conformational flexibility of PDIA1 that allows the formation of an interdomain disulfide bond. The a´ active site act then as a crossing point to redirect electrons to the ER downstream oxidases or back to client proteins. The two active sites of PDIA1 work cooperatively as an interdomain redox relay that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. Moreover, this mechanism reveals a new rational for shutting perpetuity for this down oxidative protein folding under ER redox imbalance or when the levels of unfolded proteins and folding intermediates exceed the folding capacity of the system.
- Enhancing cryopreservation of human iPSCs: Bottom-up vs Conventional freezing geometryPublication . Teodoro Duarte Garcia Morais, Fernando Jorge; El-Guendouz, Soukaina; Neves, Rafaela; Duarte, Andreia; Rodrigues, Miguel A.; Pinho Melo, EduardoInduced pluripotent stem cells (iPSCs) hold large potential on regenerative medicine due to their pluripotency and unlimited self-renewal capacity without the ethical issues of embryonic stem cells. To provide quality-controlled iPSCs for clinical therapies, it is essential to develop safe cryopreservation protocols for long-term storage, preferable amenable for scale-up and automation. We have compared the impact of two different freezing geometries (bottom-up and conventional radial freezing) on the viability and differentiation capability of human iPSCs. Our results demonstrate that the bottom-up freezing under optimized conditions significantly increases iPSCs viability, up to 9% for the cell membrane integrity and up to 21% for the cell metabolic state, compared to conventional freezing. The improvement achieved for bottom-up versus conventional freezing was maintained after scale-up from cryogenic vials to 30 mL bags, highlighting the method’s potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their future use in regenerative medicine.
- Enhancing cryopreservation of human induced pluripotent stem cells: bottom‐up versus conventional freezing geometryPublication . Teodoro Duarte Garcia Morais, Fernando Jorge; El-Guendouz, Soukaina; Neves, Rafaela; Duarte, Andreia; Rodrigues, Miguel A.; Pinho Melo, EduardoInduced pluripotent stem cells (iPSCs) hold large potential in regenerative medicine due to their pluripotency and unlimited self-renewal capacity without the ethical issues of embryonic stem cells. To provide quality-controlled iPSCs for clinical therapies, it is essential to develop safe cryopreservation protocols for long-term storage, preferably amenable to scale-up and automation. We have compared the impact of two different freezing geometries (bottom-up and conventional radial freezing) on the viability and differentiation potential of human iPSCs. Our results demonstrate that bottom-up freezing under optimized conditions significantly increases iPSC viability, up to 9% for cell membrane integrity and up to 21% for cell metabolic state, compared to conventional freezing. The improvement achieved for bottom-up versus conventional freezing was maintained after scale-up from cryogenic vials to 30 mL bags, highlighting its potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their application in regenerative medicine.