Frontiers of Biological Energetics. Electrons to Tissues

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APEX-fused POI is overexpressed in live cells and targeted to the compartment of interest, such as the mitochondrial intermembrane in this example. The typical experimental procedure involves incubating cells with biotin-phenol 30 min, 0. B BioID method. BioID experiment typically requires 18 h or longer periods of time to achieve significant biotinylation of the proteins. TurboID and miniTurboID have significantly improved the biotinylation efficiency 10 min — 2 h with little difference in labeling output. Inset shows the underlying chemistry.

Because of the short half-life and ectopic nature of the phenoxyradical Rhee et al. Thus, multiple cross comparisons may be required to build up a complete picture of the interactome. Soybean peroxidase is known to generate spontaneous reactive oxygen species under certain conditions, and hence APEX could confer context specific changes to cellular redox levels that are difficult to address Kimura and Kawano, Control for the peroxide is an intrinsic challenge of the peroxidase-based platform.

The bioactivity of the phenoxy-radical-precursor that cells are treated with for significant dose 0.

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BioID uses an engineered promiscuous E. However, in the biological milieu, biotin-AMP is ephemeral and has a diffusion distance of around 10 nm Kim et al. Recently, there have been some extensions to this method to improve the context dependence, including split-BioID De Munter et al. Critically, two different split proteins have been reported, testifying to the versatility of BioID. Finally, BioID has recently been coupled with affinity purification MS, using Strep-II tag to allow more quantitative analysis of interaction distances across large complexes Liu et al.

This issue was recently overcome by engineering ligases with heightened kinetic proficiencies. Although, little difference in the labeled proteins detected was observed between the two conditions in cultured cells, several important applications were shown in model organisms.

For instance, the embryonic development of C. Thus, a rapid labeling strategy is required to enable sufficient build-up of labeled protein in the embryonic stages. Although this question has not been systematically addressed, and despite ATP being a critical component of the cell, it is known that ATP levels are fluxional Imamura et al. Evidence for this was provided as TurboID-expressing worms were developmentally delayed, although this was not observed in miniTurboID-expressing worms; the reasons for these differences are unknown.

It is possible that split TurboID would obviate some of these issues. Another recent innovation aimed at mapping the cellular interactome exploits the enzymatic formation of acylphosphate intermediates on ubiquitin-like small protein domains, namely, prokaryotic ubiquitin-like protein PUP Pearce et al. Since Nedd8 is an endogenous modification process in cultured mammalian cells, evaluation of the specificity of the tagging process is more complex than PUP.

An extension to the Nedd8 approach has also been applied to identification of ligand—protein interactions. Cell-based studies focused on the interactome of membrane proteins including CD28 Liu et al. Critically, modification by PUP was exclusively at lysine. Furthermore, several new interactions were identified, and these interactors were dependent on the presence of the CD28 C-terminal tail Figure 3C. The experiments were carried out over a period of 24—36 h, timescale to similar to those used in Bio-ID. However, it is noteworthy that acylphosphate half-lives are variable in solution and biological systems being dependent likely on enzymatic and metal-catalyzed hydrolysis, to name two variables Di Sabato and Jencks, ; Parvez et al.

Thus, factors affecting longevity, diffusivity and off-target interactions are likely different between the methods. Interestingly, although both Bio-ID and PafA are ATP-hydrolyzing proteins whose kinetics are readily assessable in vitro , these have not been quantitatively compared. These differences clearly affect several biophysical aspects of the reactive intermediates, including: 1 the diffusion properties of the two molecules diffusion distances decrease rapidly in cells as a function of size Parvez et al.

Finally, it is noteworthy that immediately post synthesis, PafA is able to activate PUP and label interacting proteins. Furthermore, because the dwell time of CD28 is relatively short during its maturation compared to its final localization Stoops et al. APEX, with its faster labeling kinetics and small molecule substrate would likely be able to ID more potential interacting proteins especially from locales where CD28 does not ultimately reside , especially if used in conjunction with inhibitors. Other reactive-molecule-generation methods have recently been disclosed that function similarly to those discussed above, such as reactive N-arylation by N-acyl transferases Kleinpenning et al.

The logic of these extensions goes that a more detailed idea of the interaction region can be gained using such strategies. However, in order for such experiments to work well, the resolution would have to be less than the size of a protein domain 2—5 nm—a scenario unlikely to be easily achievable based on reported diffusion distances Parvez et al. There would also have to be assumptions that all residues react equally with these high-energy probes. Protein localization is a critical parameter governing protein function. For instance, many proteins gain new associations, or functions upon translocation leading to important cellular responses.

In some cases, the amount of translocation or partitioning of a protein between different organelles can be minimal. Whether such small fold changes could be reliably detected by APEX localization studies and similar methods, in our opinion, remains to be conclusively proven. The question of where proteins localize has been studied traditionally by immunofluorescence IF and fractionation. Both methods are powerful and often give consistent outcomes.

These methods are ostensibly quantitative and so in principal can give an idea of relative amounts of protein in one locale over another and can measure even quite small changes. However, it is worth remembering that traditional methods tend to suffer from limited spatial resolution and low sensitivity. This is for a number of reasons. Both methods suffer from intrinsic artifacts: for IF fixing can affect protein localization antigen presentation, whereas use of fluorescent proteins can affect target protein localization; during fractionation proteins can leak from membranes or there can be contamination from unintended structures.

Thus, in our opinion at least, perhaps the biggest improvement that reactive labeling methods bring to localization studies is the ability to couple an unambiguous readout MS to stringent tagging protocol that is strongly spatially restricted. There is estimated to be , protein-protein interactions PPIs in human cells, although this number reflects only a fraction of a percent of the total number of possible pairwise interactions Stumpf et al. Many of these PPIs are robust, with relatively long half-lives and K d 's in the nanomolar range. These methods have benefits in that they can be carried out in native cells, tissue, etc.

However, requirement for lysing of the cells can introduce artifacts due to dysregulation of cellular compartmentalization, allowing interactions that do not happen in the cell to occur Fu et al. The reaction products of cross-linking experiments are also complex aggregates that require extensive verification and typically excellent antibodies that have been rigorously validated.

Even though post-lysis associations are minimized by cross linking, there is little information offered concerning where in the cell this association occurs. This can be addressed by imaging experiments. Fluorescence colocalization of FP, or otherwise tagged proteins, or immunofluorescence has been used to visualize associations in live cells Pedley and Benkovic, , as has FRET Kenworthy, and similar methods Coffey et al.

The use of proximity ligation Fredriksson et al. This method is signal amplifying, and hence very sensitive. There are numerous genetic methods to probe PPIs. The most commonly investigated method is the yeasthybrid Y2H assay Vidal and Fields, This method uses a split transcription factor one terminus of which is fused to a bait protein, and the other terminus of which is typically fused to a series of test proteins. Pairwise combinations of the bait and each test construct are expressed in yeast. When the bait and a test protein interact, the split transcription factor is able to form a viable protein, and typically drives transcription of a gene required for survival, such that only cells expressing proteins that interact with the test protein survive.

Aside from the requirement to use ectopic protein and the fact that the native proteins are not used, criticism has been levied at this method because yeast is not a similar environment to human cells in terms of complexity, organelle structure and the posttranslational modifications it is capable of. Interactions must also happen in the nucleus. Furthermore, many Y2H methods are based off a 2 micron-plasmid system Chan et al. However, false positives are clearly not as detrimental as false negative, which are also abundant due to incomplete coverage of screening libraries, incomplete expression and poor folding.

Y2H has been extended to mammalian cells, where more complex modifications are possible, but many of the same issues remained, and the library generations are arguably more complex. Non-allelic non-complementation is a screening method that looks for unexpected non-complementation i. The likely explanation for such an effect is that proteins reside in the same pathway, and commonly these proteins form a complex that is so depleted in the double heterozygote complementation is not possible.

Although this is clearly an indirect assay, it has proven very informative and variations of this assay have been used to uncover interesting aspects of cancer biology Davoli et al. Aside from these in-cell-relevant experiments, phage display has also been used for HT-protein protein interaction screening Gibney et al. This method is of course sensitive and accurate. However, it lacks the ability to be employed in cells Kokoszka and Kay, REX technologies developed by our laboratory were ultimately aimed at studying the signaling function of reactive electrophilic species RES in living systems with individual-protein specificity and in precise space and time Figure 4 Fang et al.

The method uses custom-designed bi-functional small-molecule probes [such as Ht-PreHNE for controlled release of a native electrophile 4-hydroxynonenal HNE ]. One terminus of the probe binds HaloTag irreversibly by virtue of a pendant alkylchloride function. The other end of the bi-functional probe delivers a payload of a specific reactive electrophilic species, e. Figure 4. A T-REX electrophile delivery. A functional Halo-POI fusion protein is expressed either transiently or stably in live cells, worms, or larval fish. Provided the resulting substoichiometric RES-modification of the POI is sufficient to elicit either gain-of-function or dominant-loss-of-function signaling responses, T-REX presents a unique means to directly link target-engagement to function.

When the alkyne-modified version of the probe is used, the magnitude of measured responses can be quantitatively correlated with the POI-target-occupancy by fluorescence-gel-based analysis following Click coupling of the alkyne-functionalized-RES-modified-POI with an azido-fluorophore. B G-REX profiling. Without fusing Halo to any proteins, G-REX approach that allows for user-defined time-, dose-, and locale-controlled release of a specific RES is set to directly capture localized native sensors i.

We discuss below two different REX technologies, as well as potential or yet-unnoticed shortcomings of the method. Using T-REX, HNEylation, at individual protein-specific levels, has been shown to impact numerous critical signaling subsystems and pathway intersections, including ubiquitination Zhao et al. G-REX vide infra Zhao et al. We have also identified point mutants that are enzymatically or functionally active but do not sense the RES delivered under T-REX conditions Long et al.

Notably, these mutants are also refractory to downstream signaling changes induced upon T-REX Long et al. T-REX has found application to several model organisms, such as C. G-REX has as yet not been so applied. It was noted that in these systems, expression of the transgenes was similar to that of the endogenous proteins Long et al.

Satisfyingly in both cases, delivery and downstream signaling was observed in zebrafish similarly to cell culture. However, because of the implicit requirement of UV-light that is poorly tissue-penetratable, whole organism studies with T-REX on, for instance, mice or adult fish, are not yet possible. This current limitation would not restrict use in certain organs like the brain or blood, however. G-REX Figure 4B was established to address limitations underlying with the existing RES-sensor profiling strategies, which rely upon high doses of reactive covalent chemicals for long periods of time.

Such flooding strategies tend to incur significant off-target effects due to mass action. These approaches, although they likely achieve high occupancy and modification of multiple potential targets, also affect physiology through, for instance, perturbing cellular redox environment, and inducing stress and apoptosis. RES-permeability, intracellular distribution, metabolism, and specific subcellular redox environments, etc. HNEylated proteins are biotinylated by Click coupling with azido-biotin, precipitation, resolubilization, and streptavidin enrichment followed by mass spectrometry.

By contrast, G-REX is not intended to study downstream signaling. Several biochemical methods further document these findings.

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Thus G-REX is an unusual strategy in that it is a global method that aims to achieve only low-occupancy on-target proteins Liu et al. Its spatial resolution is currently unknown, although HaloTag itself has been successfully localized to specific subcellular compartments. G-REX has several method-specific limitations. Such issues can be circumvented by repeating experiments numerous 3 or more times and further integrating quantitative proteomics such as SILAC Ong et al.

For lipid-derived electrophiles LDEs , alkyne tagging is minimally if at all invasive, although alkynylated versions of many drugs have been successfully deployed for successful target ID Wright and Sieber, ; Parker et al. Radioisotope tagging or antibody affinity methods present alternatives to alkyne. The effects of HaloTag protein on cellular functions are not clearly known, although HaloTag has been applied to numerous systems with little negative effects reported England et al.

This outcome indicates that the origin of HNE-liberation is not particularly relevant to sensing. However, we are still unsure whether solvent cage entry is rate-limiting for POI-modification, and if reorganization may cause unanticipated issues that affect efficacy of for instance, POI RES-sensing, or conformational properties of ligand and POI. All sensors assayed were found to be uniquely HNE-sensitive. Third, photocaged probes, such as Ht-PreHNE, may be subject to inherent biases intrinsic concern for any small-molecule probe.

Beyond deploying various REX-technical controls and hypomorphic sensing-defective functional mutants Long et al. Improved photocaging strategies are presently being undertaken to further limit the possibility of artifacts. However, in these methods, the photocaged cannot be completely washed away, due to having no probe-anchoring device, such as Halo.


Organelle specificity has been instead achieved by chemical means, such as fusing triphenylphosphine to direct the probe to the mitochondria Wagner et al. The probe concentrations and those of the released lipids are difficult to normalize under these systems, and are likely not readily tunable or comparable between different cells.

When using an ectopic protein, as the ectopic protein expression can be calculated, and the amount of precursor on the protein can also be assessed, these values are much more readily normalized. To some extent, the adverse effects of the excess probe and the uncaged species can be circumvented by irradiating specific sections of a cell. However, this approach is restricted to single or a few cell-based analyses.

Improvement and further expansion should be built on our better understanding and appreciation of where the field currently is in terms of limitations that it faces and successes it has had, on our conscious and responsible use of methods and understanding of systems to apply them, and on having a firm idea of where the field is going. We strongly believe that chemical biology has the ability to deeply probe complex biological questions but our progress is hampered by reliance on unrealistic models and analogy to former biochemical studies.

Using the most relevant model systems will be an enabling step forward in successfully tackling the important problems unsolvable by traditional genetics and biochemistry. XL contributed to the creation of all figures and tables as well as manuscript editing and formatting.

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Cellular labelling favours unfolded proteins.