Chemical zymogens for the protein cysteinome

Montasell, Mireia Casanovas; Monge, Pere; Carmali, Sheiliza; Loiola, Lívia M.D.; Andersen, Dante Guldbrandsen; Løvschall, Kaja Borup; Søgaard, Ane Bretschneider; Kristensen, Maria; Pütz, Jean Maurice; Zelikin, Alexander N.

doi:10.1038/s41467-022-32609-1

Cited by 21 publications

(28 citation statements)

References 48 publications

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“…The faster reaction reported herein will be more favorable to pyridinic end-cap disulfide bonds but can also occur with backbone disulfides. Self-immolative poly(disulfide) materials based on lipoic acid have been shown to depolymerize in an alkaline environment or by addition of reductive thiols, reforming their monomers in high yields [ 51 , 52 , 53 ]. However, electrochemically induced degradation of self-immolative disulfides, or SIPs in general, is a novel approach, let alone exploitation of its catalytic nature.…”

Section: Discussionmentioning

confidence: 99%

Electrocatalytic Depolymerization of Self-Immolative Poly(Dithiothreitol) Derivatives

2022

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We report the use of electrogenerated anthraquinone radical anion (AQ•−) to trigger fast catalytic depolymerization of polymers derived from poly(dithiothreitol) (pDTT)—a self-immolative polymer (SIP) with a backbone of dithiothreitols connected with disulfide bonds and end-capped via disulfide bonds to pyridyl groups. The pDTT derivatives studied include polymers with simple thiohexyl end-caps or modified with AQ or methyl groups by Steglich esterification. All polymers were shown to be depolymerized using catalytic amounts of electrons delivered by AQ•−. For pDTT, as little as 0.2 electrons per polymer chain was needed to achieve complete depolymerization. We hypothesize that the reaction proceeds with AQ•− as an electron carrier (either molecularly or as a pendant group), which transfers an electron to a disulfide bond in the polymer in a dissociative manner, generating a thiyl radical and a thiolate. The rapid and catalytic depolymerization is driven by thiyl radicals attacking other disulfide bonds internally or between pDTT chains in a chain reaction. Electrochemical triggering works as a general method for initiating depolymerization of pDTT derivatives and may likely also be used for depolymerization of other disulfide polymers.

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Section: Discussionmentioning

confidence: 99%

Electrocatalytic Depolymerization of Self-Immolative Poly(Dithiothreitol) Derivatives

2022

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“…The main difference, and therefore the main innovative aspect of the EAR signaling, is in the mechanism of signal transduction: the 1,6-benzyl elimination of a self-immolative linker. Signal transduction design presented herein extends our work on the protein cysteinome 37 and the released secondary messenger is an amino acid L-Cys, for activation of the disulfide-based chemical zymogens. Thiol inhibition and diffusion studies validated the receptor-like mechanism of signal transduction mediated by the EAR molecules.…”

Section: Resultsmentioning

confidence: 72%

“…To mimic this, we used a cysteine protease, papain. The activity of this enzyme is blocked by a chemical modification of the catalytic thiol group in the active site, namely by conversion into a disulfide 36 , 37 . The catalytic activity of the chemical zymogen is restored via thiol-disulfide exchange with another thiol-containing molecule—thus presenting an opportunity to achieve chemically triggered activation of enzymatic catalysis 37 .…”

Section: Resultsmentioning

confidence: 99%

“…The activity of this enzyme is blocked by a chemical modification of the catalytic thiol group in the active site, namely by conversion into a disulfide 36 , 37 . The catalytic activity of the chemical zymogen is restored via thiol-disulfide exchange with another thiol-containing molecule—thus presenting an opportunity to achieve chemically triggered activation of enzymatic catalysis 37 . This notion suggested that the secondary messenger released by the receptor upon its exofacial activation must be a thiol-containing solute.…”

Section: Resultsmentioning

confidence: 99%

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Transmembrane signaling by a synthetic receptor in artificial cells

et al. 2023

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Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

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“…In our own recent work, we approached the protein cysteinome with a specific purpose of designing chemical zymogens, that is, reversibly deactivated enzymes. [15] The highest achievement of our work was that we designed disulfide-based chemical zymogens that could be reactivated by another thiol-containing protein. Based on this finding, we engineered a process whereby the two proteins perform cross-(de)activation in what we termed a zymogen exchange reaction.…”

Section: Introductionmentioning

confidence: 99%

Chemical Zymogens: Specificity and Steroidal Control of Reactivation

2023

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Activating and masking enzymatic activity on demand is of the highest importance in nature. It is achieved by chemical interconversion of enzymes and the corresponding zymogens through, for example, proteolytic processing or reversible phosphorylation, and affords on-demand activation of enzymes, controlled in space and/or time. In stark contrast, examples of chemical zymogens are very few, and in most cases these are based on disulfide chemistry, which is largely indiscriminate as to the nature of the activating thiol. In this work, we address an outstanding challenge of specificity of reactivation of chemical zymogens. We achieve this through engineering affinity between the chemical zymogen and the activator. Additional, higher-level control over zymogen reactivation is installed in a nature-mimicking approach using steroidal hormones. Taken together, the results of this study take a step towards establishing the specificity of reactivation of synthetic, chemical zymogens. We anticipate that the results of this study will contribute significantly to the development of chemical zymogens as tools for diverse use in chemical biology and biotechnology.[a] M.

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Chemical zymogens for the protein cysteinome

Cited by 21 publications

References 48 publications

Electrocatalytic Depolymerization of Self-Immolative Poly(Dithiothreitol) Derivatives

Electrocatalytic Depolymerization of Self-Immolative Poly(Dithiothreitol) Derivatives

Transmembrane signaling by a synthetic receptor in artificial cells

Chemical Zymogens: Specificity and Steroidal Control of Reactivation

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