Biology subjects

Melo, E. P.

Publications and source records attributed to Melo, E. P..

3 recordsLinked to original sources

bioRxiv2024

Enhancing cryopreservation of human iPSCs: Bottom-up vs Conventional freezing geometry

Induced 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 methods 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.

cell biology↗

bioRxiv2023

A conformational-dependent interdomain redox relay at the core of Protein Disulfide Isomerase activity

Protein 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 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.

biochemistry↗

bioRxiv2016

TriPer, an optical probe tuned to the endoplasmic reticulum, tracks H2O2 consumption by glutathione

The fate of H2O2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic {beta}-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H2O2 signal and eroded {beta}-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.\n\nSignificance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathiones role ER H2O2 metabolism.\n\nHere we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.\n\nThis study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

cell biology↗