1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein

  1. Mikel Garcia-Marcos  Is a corresponding author
  2. Kshitij Parag-Sharma
  3. Arthur Marivin
  4. Marcin Maziarz
  5. Alex Luebbers
  6. Lien T Nguyen
  1. Boston University School of Medicine, United States
Tools and Resources
  • Cited 3
  • Views 1,338
  • Annotations
Cite this article as: eLife 2020;9:e60155 doi: 10.7554/eLife.60155

Abstract

Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3 and 4 (and their corresponding supplements).

Article and author information

Author details

  1. Mikel Garcia-Marcos

    Department of Biochemistry, Boston University School of Medicine, Boston, United States
    For correspondence
    mgm1@bu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9513-4826

  2. Kshitij Parag-Sharma

    Department of Biochemistry, Boston University School of Medicine, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3638-0941

  3. Arthur Marivin

    Department of Biochemistry, Boston University School of Medicine, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Marcin Maziarz

    Department of Biochemistry, Boston University School of Medicine, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Alex Luebbers

    Department of Biochemistry, Boston University School of Medicine, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7733-4250

  6. Lien T Nguyen

    Biochemistry, Boston University School of Medicine, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institute of General Medical Sciences (R01GM136132)

  • Mikel Garcia-Marcos

American Cancer Society (PF-19-084-01-CDD)

  • Marcin Maziarz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Publication history

  1. Received: June 18, 2020
  2. Accepted: September 15, 2020
  3. Accepted Manuscript published: September 16, 2020 (version 1)
  4. Version of Record published: September 24, 2020 (version 2)

Copyright

© 2020, Garcia-Marcos et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,338
    Page views
  • 220
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organisms

Further reading

    1. Biochemistry and Chemical Biology

    De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition

    Erich Stefan et al.
    Research Article Updated

    ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

    1. Biochemistry and Chemical Biology

    Therapeutic inhibition of keratinocyte TRPV3 sensory channel by local anesthetic dyclonine

    Qiang Liu et al.
    Research Article Updated

    The multimodal sensory channel transient receptor potential vanilloid-3 (TRPV3) is expressed in epidermal keratinocytes and implicated in chronic pruritus, allergy, and inflammation-related skin disorders. Gain-of-function mutations of TRPV3 cause hair growth disorders in mice and Olmsted syndrome in humans. Nevertheless, whether and how TRPV3 could be therapeutically targeted remains to be elucidated. We here report that mouse and human TRPV3 channel is targeted by the clinical medication dyclonine that exerts a potent inhibitory effect. Accordingly, dyclonine rescued cell death caused by gain-of-function TRPV3 mutations and suppressed pruritus symptoms in vivo in mouse model. At the single-channel level, dyclonine inhibited TRPV3 open probability but not the unitary conductance. By molecular simulations and mutagenesis, we further uncovered key residues in TRPV3 pore region that could toggle the inhibitory efficiency of dyclonine. The functional and mechanistic insights obtained on dyclonine-TRPV3 interaction will help to conceive therapeutics for skin inflammation.

    1. Biochemistry and Chemical Biology

    In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases

    Molly C Sutherland et al.
    Research Article

    Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.