Activity-based directed evolution of a membrane editor in mammalian cells

Tei, Reika; Bagde, Saket R.; Fromme, J. Christopher; Baskin, Jeremy M.

doi:10.1038/s41557-023-01214-0

Cited by 14 publications

(22 citation statements)

References 72 publications

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“…Because LOVPLD exhibited similar levels of enzymatic activity on these organelles, as revealed by transphosphatidylation-based IMPACT (Figure e) and because transphosphatidylation tracks well with hydrolysis activity in PLD PMF , , we can infer that hydrolysis activity, i.e., PA production, should also be similar in all organelles. Therefore, we propose that the lack of PA accumulation on certain organelle membranes is likely due to differential rates of PA consumption on and clearance from these organelles.…”

Section: Resultsmentioning

confidence: 68%

“…The interaction of PA with LKB1 has been suggested to be important, and we had previously observed that cells expressing superPLDs showed slightly elevated global levels of p-AMPK. 12,20 Using an expanded palette of organelle-targeted LOVPLDs whose expression was well tolerated, we first confirmed that PA production on mitochondria, the ER, lysosomes, or the Golgi complex indeed induces LKB1 recruitment.…”

Section: ■ Discussionmentioning

confidence: 71%

“…We assessed the activity of LOVPLD variant P (hereafter referred to simply as LOVPLD) relative to the dimerizationbased optogenetic superPLD low , which was previously optimized for mammalian cell-based membrane editing applications. 20 Whereas the latter achieved a 1.6-fold increase in IMPACT labeling upon blue light activation, LOVPLD reached a similar level of maximum activity but with a 6.4fold increase in IMPACT labeling, with the increase in signalto-noise coming entirely from an almost 4-fold decrease in offstate activity (Figure 1d). We verified the PM localization of the Lyn 10 -anchored LOVPLD and catalytically dead LOVPLD (dLOVPLD) using confocal microscopy and further showed that IMPACT labeling in LOVPLD-expressing cells required blue light illumination (Figure 1e).…”

Section: Lov Domain Insertion Into Superpld Renders Itmentioning

confidence: 89%

“…Most recently, we substantially improved the catalytic activity of optoPLD to yield a family of superactive PLDs (superPLDs) by increasing its stability in the reductive cytosolic environment via mammalian cell-based directed evolution. 20 SuperPLDs catalyze both hydrolysis and transphosphatidylation with significantly higher efficiency than wild- type PLD PMF , indicating their potential as highly versatile phospholipid editors within living cell membranes. However, optogenetic superPLDs exhibited increased background activity in the absence of the light trigger, i.e., without membrane recruitment, limiting their temporal resolution and causing unwanted, off-target effects.…”

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling

Li,

Tei,

Uematsu

et al. 2024

ACS Cent. Sci.

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Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light−oxygen−voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.

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Section: Resultsmentioning

confidence: 68%

Section: ■ Discussionmentioning

confidence: 71%

Section: Lov Domain Insertion Into Superpld Renders Itmentioning

confidence: 89%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling

Li,

Tei,

Uematsu

et al. 2024

ACS Cent. Sci.

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Optogenetic Strategies for Optimizing the Performance of Phospholipids Biosensors

Yao,

Lou,

Jin

et al. 2024

Advanced Science

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High‐performance biosensors play a crucial role in elucidating the intricate spatiotemporal regulatory roles and dynamics of membrane phospholipids. However, enhancing the sensitivity and imaging performance remains a significant challenge. Here, optogenetic‐based strategies are presented to optimize phospholipid biosensors. These strategies involves presequestering unbound biosensors in the cell nucleus and regulating their cytosolic levels with blue light to minimize background signal interference in phospholipid detection, particularly under conditions of high expression levels of biosensor. Furthermore, optically controlled phase separation and the SunTag system are employed to generate punctate probes for substrate detection, thereby amplifying biosensor signals and enhancing visualization of the detection process. These improved phospholipid biosensors hold great potential for enhancing the understanding of the spatiotemporal dynamics and regulatory roles of membrane lipids in live cells and the methodological insights in this study might be valuable for developing other high‐performance biosensors.

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Systems and Methods for Continuous Evolution of Enzymes

Chen,

Zhang,

Đelmaš

et al. 2024

Chemistry A European J

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Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions.

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Activity-based directed evolution of a membrane editor in mammalian cells

Cited by 14 publications

References 72 publications

Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling

Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling

Optogenetic Strategies for Optimizing the Performance of Phospholipids Biosensors

Systems and Methods for Continuous Evolution of Enzymes

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