Development of proximity proteinlabeling
Hiroyasu Nakao
2021/10/07

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Contents・Introduction- proximity protein labeling- BioID, APEX・Microenviromental mapping・Target ID
・Summary2

Introduction

Visible-light-driven catalysis has a tremendous impact on chemical synthesis,
nanotechnology, energy and biological sectors.
O. Fedeyi, et al. Nat. Rev. Chem, 2021, 5, 322.

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Proximity protein labelling✔ Protein-protein interactions (PPIs), in which many proteins interact with otherproteins, are important.
✔ Weak PPIs have until remained almost undetectable.
✔ Proximity protein labeling is one way to obtain information about PPIs.
D. Macmillan, et al. Chem. Soc. Rev. 2021, 50, 2911.

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What is proximity protein labeling?
✔ Proximity labelling is the localization ofa small molecule or protein that cangenerate a reactive intermediate.
✔ Reactive intermediates cross-link withproteins.
✔ Extracted proteins can be analyzed byimmunoblotting and chemoproteomic
analysis
Key issue✔ labeling radius✔ labeling time
D. Macmillan, et al. Chem. Soc. Rev. 2021, 50, 2911.

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History

http://zoulab.org/Research_Proteomics.html 6

BioIDBiotinoyl-50AMP (BioAMP) is produced by thecombination of biotin and ATP.
BioAMP is retained in the active site of BirA*.
BirA* : biotin ligase
When BioAMP is released from BirA, it reacts withsurrounding lysine residues.
D. Macmillan, et al. Chem. Soc. Rev. 2021, 50, 2911.

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APEX
Iron(Ⅲ) is oxidized by hydrogen peroxide tobecome iron (IV).
Water and oxoferyl (FeIV) radical cations aregenerated via peroxo iron species.
This species is then reduced by phenol to neutral
oxoferryl to produce phenoxyl radicals.
Phenoxy radicals crosslink with tyrosine andnucleic acids.
D. Macmillan, et al. Chem. Soc. Rev. 2021, 50, 2911.

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Protein proximity labeling
MethodSubstrates

Labeling
timestrengthlimitations

APEX

APEX,APEX2

Biotin phenol ≦1 min(BP), H2O2

High temporal resolution
High activity

H2O2 toxicity
BP permeability

BioID

BioID2

Biotin, ATP

Non-toxic
stable at higher
temperatures

Poorly reactive
Low temporal control
Wide labeling radius

18 hAirID

3hTurboID≦ 10 min Highest activity

cf. Takahashisan’s proposal seminar

9Contents・Introduction- proximity protein labeling- BioID, APEX・Microenviromental mapping・Target ID
・Summary10

Microenvironment mapping

catalyst✔ tolerant of aqueous conditions✔ conjugate to antibodies, DNA, small ligand✔ selectively activate chemical probes at a diffusionlimited ratechemical probe✔ activated within 1 nm of the catalyst radius✔ not to undergo long-range diffusion afteractivation
11D. Macmillan et al. Science. 2020, 367, 1091.

Carbene

Carbenes readily cross-link with C–H bonds found in all biomolecules [(k) = 3.1 × 109 s−1]
12D. Macmillan et al. Science. 2020, 367, 1091.

Carbene

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D. Macmillan et al. Science. 2020, 367, 1091.

Photocatalyst✔ [Ir(dFCF3ppy)2(dtbbpy)]PF6(ET = 60.1 kcal/mol) is the best photocatalyst.
✔ Iridium photocatalyst(3) aimed at biomolecular applications did not affect ability
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to remove nitrogen.
D. Macmillan et al. Science. 2020, 367, 1091.

Dexter energy transfer vs Forster energy transfer✔ No overlap was observed between the absorption spectrum of 1 andthe emission spectrum of 2.
✔ It was suggested that it was a Dexter energy mechanism rather thana Forster energy transfer.

15D. Macmillan et al. Science. 2020, 367, 1091.

Protein labeling✔ No background reaction occurred.
✔ Turning the light source on or off affords fine temporal control over the labeling process.
16D. Macmillan et al. Science. 2020, 367, 1091.

Selective proximity protein labeling✔ EGFR2/VEGFR2 was selectively labeled when irradiated with 450 nm light.

✔ When CD45-targeted μMap was performed on Jurkat cells, light- and time-dependent
protein biotinylation was observed.
17D. Macmillan et al. Science. 2020, 367, 1091.

μMAP proteomics
Could μMap differentiate between spatially separated microenvironmentson the same cell membrane?

✔ The ability of μMap discriminate between unrelated microenvironments.

18D. Macmillan et al. Science. 2020, 367, 1091.

PD-L1
They investigate the proximal protein interactome of PD-L1 in B cells.

✔ It was found that CD30 (a member of the tumor necrosis factor receptor family) and CD300A(an immunosuppressive receptor), may have a new interaction due to significant enrichment.
✔ Potential of mMapping provides new insights with respect to the microenvironments of19D.
Macmillan
etal.
Science.
2020,367,1091.
checkpoint proteins.

Intercellular communication✔ Understanding intercellular communication is important.
✔ Does biotinylation of the surface of APCs expressing PD-L1 (cis labeling) lead
not only to biotinylation of T cells at adjacent synapses (trans labeling)?
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D. Macmillan et al. Science. 2020, 367, 1091.

Intercellular communication
Trans labeling

PDL1–targeted μMap showed highselectivity for trans-labeling solely at thecis- and trans-cellular contact regions.

Cis labeling

CD45RO-targeted μMap led to selective
cislabeling on the CD45RO-expressingJurkat cells.

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D. Macmillan et al. Science. 2020, 367, 1091.

Target ID✔ Target ID remains a fundamental goal in drug discovery.
D. Macmillan et al. bioRxiv preprint.

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Design of photocatalyst

Problem
Cell permeable
Commercially available
Chemical manipulation
Retention of biological activity etc

By removing the carboxylic acidgroups, the cationic photocatalyst(Gen 2) was rendered cell permeable
D. Macmillan et al. bioRxiv preprint.

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(+)-JQ128

No labeling with inactive(-)-JQ1 enantiomer(+)-JQ-128 is a potent inhibitor of
the BET family of bromodomain
proteins (BRD2/3/4).

No labeling with inactive(+)-JQ1-G1-Ir catalyst

Labeling increases
with time due tosignal amplification
D. Macmillan et al. bioRxiv preprint.

24(+)-JQ128✔ Some BRD proteins are the most enriched.
✔ ALCAM (CD166) was also identified as being significantly enriched, but currently has noreported interaction with (+)-JQ1.
✔ CD166 was detected it was not enriched, indicating that binding may not be affected by
the stereogenic center.
D. Macmillan et al. bioRxiv preprint.

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Classical photoaffinity labelingWestern blot and chemoproteomic analysis showed that the BRD protein was not enriched.
D. Macmillan et al. bioRxiv preprint.

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desHEP-Dasatinib

Dasatinib shows anti-cancer effects
as a tyrosine kinase inhibitor✔ Interactions with several established kinases, including Src and Lyn, have been identified.
✔ They identified an off-target multidrug resistance transporter, ABCC1.
D. Macmillan et al. bioRxiv preprint.

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Dasatinib✔ Iridium photocatalyst adapted to maintainbiological function and cell permeability.
✔ Well-known kinases such as p38a, Myt1, andCSK kinases were extensively enriched.

✔ The most recent optical affinity labeling wasthat CSK, BTK and MAP were slightly enriched.

D. Macmillan et al. bioRxiv preprint.

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Paclitaxel✔ The anti-cancer mechanism of Paclitxel (Taxol) is not clear.
✔ Western blot analysis using anti-a-tubulin clearly labeled the target protein compared tofree iridium and DMSO.

✔ Tubulin isotypes aIa, bIII, bIVb, and aIc were found to be extensively labeled.
D. Macmillan et al. bioRxiv preprint.

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SCH58261
Validating the µMap Target ID
Platform with Cell Membrane Proteins.

They chose the adenosine receptor A2a(ADORA2A) as a membrane target.

× PAL with SCH58261-Dz (7) showed
low enrichment of ADORA2A.
✔ Photocatalyst labeling withSCH58261-G1(8) showed similarlyhigh levels of ADORA2A enrichment.

D. Macmillan et al. bioRxiv preprint.

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Summary✔ A new mapping technology has been developed to replace APEX and BioID.
✔ μMap enables exploration of protein-protein interactions andintercellular communication.
✔ μMap targetID has allowed for the identification of multiple proteintaegets across multiple drug classes and compartments.

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