A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function Renicke et al. (2013)

A LOV2 Domain-Based Optogenetic Tool to ControlProtein Degradation and Cellular Function

Renicke et al. (2013)

Feynman Liang

Amherst College

[email protected]

April 4, 2014

Feynman Liang (AC) Renicke et al. (2013) April 4, 2014 1 / 22

Things to keep in mind throughout the talk

Tools for studying biological systems should be...

Specific

Cryptic

Generalizable

Quantitative control

Reversible

Rapid


Optogenetics

Definition

Optogenetics – the synthetic approach using genetically encoded proteinsto control protein activity by light [1]


Pop quiz!

Without looking at the rest of the data, we can predict that one bigadvantage light has over small molecules is that

Light is usually specific and does not affect endogeneous proteins

Light is not cytotoxic for most organisms so unlike small molecules itsefficacy is not limited by maximum tolerated dosage

Light-activated mechanisms are more rapid because the transport ofsmall molecules is limited by diffusion (〈x2〉 ≈ 6Dτ)


Pop quiz!

Without looking at the rest of the data, we can predict that one bigadvantage light has over small molecules is that

Light is usually specific and does not affect endogeneous proteins

Light is not cytotoxic for most organisms so unlike small molecules itsefficacy is not limited by maximum tolerated dosage

Light-activated mechanisms are more rapid because the transport ofsmall molecules is limited by diffusion (〈x2〉 ≈ 6Dτ)


What do plants have to offer?

Figure 1: Arabidopsis thaliana [2]


Phototropism

Figure 2: Summary of plant phototropism [3]


Light-Oxygen-Voltage (LOV) photosensors

Figure 3: Left: Avena sativa (oat) phototropin1 LOV2 (AsLOV2) in dark, Right:Partial proteolysis of AsLOV2 by chymotrypsin digestion [4]


A. thaliana phototropin1 LOV2 forms a coiled-coil dimer

Figure 4: Structure of A. thaliana LOV2 (AtLOV2) dimer in dark (RCSB 4HHD)[5]


Ornithine decarboxylase (ODC) degron

Figure 5: Dimerization with antizyme(AZ) causes degradation of ODC [6]

Ubiquitin-independentdegradation

Mechanism conserved acrossvertebrates, yeasts, and plants

Degradation domain atC-terminus (cODC)


Question time!

The last slide suggests that a light-activated cODC degron is likely topossess which of the following qualities?

Specific

Cryptic

Generalizable


Reversible

Rapid


Question time!

The last slide suggests that a light-activated cODC degron is likely topossess which of the following qualities?

Specific

Cryptic

Generalizable


Reversible

Rapid


Development of the photosensitive degron (psd) module

Figure 6: A: diagram of degradation process, B: construct screening using redflourescent protein (RFP) indicator


Are you paying attention?

Which of the constructs do you think the authors christen as the psdmodule and use for the remainder of the experiment?

a

b

c

d

e


Are you paying attention?

Which of the constructs do you think the authors christen as the psdmodule and use for the remainder of the experiment?

a

b

c

d

e


Degradation is effective, cryptic, and reversible

Figure 7: psd module causes reversible light-activated degradation of proteins


Degradation can be quantitatively controlled

Figure 8: Quantitative control is achieved by varying light intensity


Lets stop and take stock...

Based on what we’ve seen so far, the psd module possesses (is likelyto possess) which of the following qualities?

Specific

Cryptic

Generalizable


Reversible

Rapid? Depends on definition (t1/2 ≈ 25 min).


Lets stop and take stock...

Based on what we’ve seen so far, the psd module possesses (is likelyto possess) which of the following qualities?

Specific

Cryptic

Generalizable


Reversible

Rapid? Depends on definition (t1/2 ≈ 25 min).


Cell cycle control

Figure 9: Photocontrol of Cdc28 regulators


Conditional protein inhibition

Figure 10: Light-induced degradation of an assortment of genes


Compartmentalized ODE modeling

Figure 11: In-silico modeling of steady state concentrations, pp1 ∝ promoterstrength


Is compartmentalization (i.e. forgetting all about space and onlyconsidering time) a valid simplifying assumption or should the authorshave used a PDE model?

Yes

No


Is compartmentalization (i.e. forgetting all about space and onlyconsidering time) a valid simplifying assumption or should the authorshave used a PDE model?

Yes

No


In summary...

Developed a photo-activated degradation technique (psd module)that is

SpecificCrypticGeneralizable? Only tested yeast.Quantitative controlReversibleRapid? Depends on definition (t1/2 ≈ 38 min).

Demonstrated applicability of the technique to

Yeast photographyYeast cell cycle regulationConditional degradation of various yeast genesInduced yeast cell patterning

In silico modeling suggests efficacy most sensitive to cODC degronefficiency


References

Christian Renicke et al.

A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular FunctionJ. Chem Biol. 20(4), 619–626 (2013)

Arabidopsis thaliana

Wikipedia: The Free Encyclopedia Wikimedia Foundation, Inc. 16 Oct. 2004. Web. 3 April 2004

Phototropism

Wikipedia: The Free Encyclopedia Wikimedia Foundation, Inc. 5 Feb. 2014. Web. 3 April 2004

Shannon M. Harper et al.

Structural Basis of a Phototropin Light SwitchScience 301, 1541 (2003)

Halavaty et al.

Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana.Acta Crystallogr. Sect.F 69:, 1316–1321 (2013)

Philip Coffino & Dieter H. Wolf

Degradation of Ornithine Decarboxylase, a Ubiquitin-Independent Proteosomal ProcessProteasomes: The World of Regulatory Proteolysis Ch. 16, 254–263 (2000)


The End
