Source 1primary paper2017FEBS Journal
Toolkit/PiL[D24]
PiL[D24]
Also known as: mPKM2 internal LOV2 fusion at position D24
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
PiL[D24] is an engineered mammalian pyruvate kinase M2 (PKM2) photoswitch containing an internal insertion of the Avena sativa LOV2 light-sensing domain at position D24. Illumination preserves the LOV2 photoreaction and allosterically modulates PKM2 behavior, increasing pyruvate kinase activity and cellular labeling of pyruvate from glucose.
Usefulness & Problems
Why this is useful
PiL[D24] provides optical control over a central metabolic enzyme in mammalian cells, enabling light-dependent modulation of pyruvate kinase output. This is useful for probing how acute changes in PKM2 activity affect glucose-to-pyruvate flux in cellular systems.
Problem solved
PiL[D24] addresses the problem of reversibly controlling mammalian PKM2 activity with light rather than static genetic or chemical perturbation. The reported construct links a photosensory LOV2 input to enzymatic regulation of pyruvate kinase and downstream pyruvate labeling from glucose.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Mechanisms
light-induced allosteric switchinglight-induced secondary-structure changelov2 photoreactionsecondary-structure changeTechniques
Computational DesignTarget processes
recombinationsignalingInput: Light
Implementation Constraints
PiL[D24] is a domain-fusion construct consisting of mammalian PKM2 with an internal LOV2 insertion at residue D24, using the Avena sativa LOV2 photosensory domain. Molecular dynamics simulations were used to guide design, but the provided evidence does not include construct architecture details beyond the insertion site or practical delivery and expression conditions.
The supplied evidence comes from a single 2017 study and does not describe independent replication. The available evidence does not specify illumination wavelength, dynamic range, reversibility kinetics, expression constraints, or validation beyond the reported enzymatic and cellular labeling effects.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Km change for phosphoenolpyruvate 30 %
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
Approval Evidence
1 source6 linked approval claimsfirst-pass slug pil-d24
mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24]), an engineered pyruvate kinase M2 (PKM2) variant that harbours an insertion of the light-sensing LOV2 domain from Avena Sativa
Source:
activity modulationsupports
Light exposure causes secondary structure changes in PiL[D24] that are associated with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity.
causes secondary structure changes that are associated with a 30% decrease in the Km of the enzyme for phosphoenolpyruvate resulting in increased pyruvate kinase activity after light exposure
Source:
cellular effectsupports
Expression of PiL[D24] in cells leads to a light-induced increase in labelling of pyruvate from glucose.
Expression of PiL[D24] in cells leads to light-induced increase in labelling of pyruvate from glucose.
Source:
engineered designsupports
Molecular dynamics simulations were used to guide the design of the PiL[D24] mPKM2-LOV2 fusion.
we have used molecular dynamics simulations to guide the design of mPKM2 internal light/oxygen/voltage-sensitive domain 2 (LOV2) fusion at position D24 (PiL[D24])
Source:
mechanismsupports
The LOV2 photoreaction is preserved in the PiL[D24] chimera.
The LOV2 photoreaction is preserved in the PiL[D24] chimera
Source:
proposed usesupports
PiL[D24] could provide a means to modulate cellular glucose metabolism remotely.
PiL[D24] therefore could provide a means to modulate cellular glucose metabolism in a remote manner
Source:
reversibilitysupports
The light-induced change in PiL[D24] activity is reversible upon light withdrawal.
Importantly, this change in activity is reversible upon light withdrawal.
Source:
Comparisons
Source-backed strengths
The reported variant shows light-associated secondary-structure changes together with a 30% decrease in Km for phosphoenolpyruvate and increased pyruvate kinase activity. It was also validated in cells, where expression of PiL[D24] produced a light-induced increase in labeling of pyruvate from glucose.
Ranked Citations
- 1.