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Talk:Pallag 2022 Abstract Bioblast

From Bioblast

Comments by Reviewers and Authors responses

Moreno-Sanchez Rafael and authors (2022-05-30)
  • The work described in this abstract seems relevant and novel. It might become essential for cancer cell bioenergetics, as some malignant cells show an enhanced proline metabolism. To further strengthen the findings and claims of the work, the authors may want to deal with the following observations.

1. Proline catabolism generated a sufficiently high membrane potential ...

  • A quantitative value in mV for the membrane potential would be a stronger result.
Response: indeed, calibrated safranine or rhodamine123 fluorescence signals are shown in the work (now published in https://www.mdpi.com/1422-0067/23/9/5111/htm) but the extend of alterations are too many and variable depending on conditions, thus โ€œone value fits them allโ€ principle could not be applied. Therefore, we do not indicate a quantitative value in mV for the membrane potential in the abstract, but calibrated safranine or rhodamine123 fluorescence signals will be shown in the presentation.

2. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline

  • Duroquinone is the oxidized species. You probably meant duroquinol or reduced duroquinone.
Response: indeed, duroquinone stocks have been reduced to duroquinol with borohydride and hydrochloric acid, we change this in the revised abstract; as a side note though, we never โ€œtitrateโ€ or add a calculated amount of borohydride amount needed for full duroquinone reduction, as this carries the risk of carrying over excess borohydride to the stock solution and further on to mitochondrial suspension; stock solutions are not periodically re-reduced, thus, over time, it is possible (and actually very likely) that duroquinol partially reoxidizes to duroquinone. I am only mentioning this as to raise awareness regarding the use of duroquinone/duroquinol.

3. Excess glutamate, however, could not reproduce the proline effect

  • Were these experiments carried out with some malate added, which is required to ensure full glutamate oxidation through transamination?
Response: yes, malate was always present when glutamate concentration was varied, this is detailed in the work; we have added this clarification in the revised abstract.

Version 1

2022-05-12
The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements.
NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality.
Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects.
The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.