Optogenetic control of necroptosis pathway

Oh, Teak-Jung

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https://hdl.handle.net/2142/125781 Copy

Description

Title

Optogenetic control of necroptosis pathway

Author(s)

Oh, Teak-Jung

Issue Date

2024-07-09

Director of Research (if dissertation) or Advisor (if thesis)

Zhang, Kai

Doctoral Committee Chair(s)

Zhang, Kai

Committee Member(s)

• Chen, Jie
• Chen, Lin-Feng
• Nelson, Erik

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Necroptosis RIPK3 Optogenetics Spatiotemporal control Inflammatory Lytic cell death

Abstract

Necroptosis is a form of immunogenic cell death marked by distinct cellular responses including cytokine production and plasma membrane rupture (PMR), playing a pivotal role in various pathological conditions such as inflammatory diseases and cancers. Among the key proteins in the necroptosis pathway, Receptor-Interacting Protein Kinase 3 (RIPK3) is essential for orchestrating cytokine production and PMR, which can culminate in an inflammatory response. However, the complex regulation of RIPK3 for cytokine production and PMR, as well as its role in the intercellular necroptosis pathway, has not been clearly elucidated. The innovative technique of opsin-free optogenetics has been successfully employed to dissect and control a wide range of cell signaling in live cells and animals with unprecedented spatiotemporal accuracy. As a synthetic biology approach, I have developed a blue-light-responsive module to control RIPK3-dependent necroptosis, called the light-activatable RIPK3 (La-RIPK3) system. Activation of La-RIPK3 facilitates the dissection of RIPK3-driven necroptosis by inducing the formation of a RIPK3-containing necrosome, which is crucial for cytokine production and PMR. Additionally, La-RIPK3 stimulates cytokine production, which is predominantly dependent on RIPK3 kinase activity. This activity synergistically induces immune cell migration in conjunction with PMR-mediated release of intracellular contents. Comprehensive RNA sequencing analysis reveals that RIPK3 oligomerization distinctly alters gene expression patterns, differing significantly from those induced by the treatment with pharmacological necroptosis inducers. Further investigation into necroptosis pathways revealed the threshold-like regulation of both intracellular and intercellular necroptosis pathways via La-RIPK3 activation. For intracellular necroptosis, PMR seems to require a higher threshold of RIPK3 activity compared to cytokine production. Furthermore, super-resolution imaging revealed that the morphology of RIPK3 aggregates—spherical versus rod-like shapes—may influence the efficiency of molecular interactions involved in PMR and cytokine production. Moreover, RIPK3-induced lytic cell death can propagate intercellularly, with cells surrounded by multiple necroptotic neighbors more likely to undergo PMR, suggesting a collective threshold of necroptotic signals necessary for triggering cell death propagation. RIPK3 has also been shown to exhibit dual roles in solid tumors, serving as both an anti-tumor and pro-tumor factor. In breast cancer, it was suggested that RIPK3 can benefit the survival probability of patients. With the goal of solid tumor eradication, the concluding part of the work highlights the successful application of La-RIPK3 in a triple-negative breast cancer (TNBC) model, where La-RIPK3 can be used to further applied for modulation the tumor microenvironment (TME) by inducing necroptosis in a living animal. By employing La-RIPK3 in a TNBC model, it is possible that acutely induced necroptosis can shift the TME from an immunosuppressive to an immunogenic state. This shift can be critical for enhancing the efficacy of immunotherapies in cancer treatment. Overall, this work not only expands our understanding of necroptosis pathways and its regulation but also sets the stage for future research that could transform therapeutic approaches in immune-related diseases and cancer. By leveraging the precision of optogenetics, this work offers a new paradigm for investigating necroptosis pathways and their implications in disease, making a significant contribution to the field of synthetic biology, cell death research, cancer biology.

Graduation Semester

2024-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/125781

Copyright and License Information

Copyright 2024 Teak Jung Oh

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