Q&A with JCC Fellow Yunhao Tan

June 26, 2019
Innate Immune Signaling Organelles Display Natural and Programmable Signaling Flexibility

Recent research published in Cell by JCC Fellow Yunhao Tan demonstrates the flexibility and programmability of immune signaling organelles. In this Q&A, Tan explains his work, completed with Jonathan Kagan at Harvard Medical School.

Q) Taking a broad view, how does your work fit in with the immune system as a whole?

The mammalian immune system is composed of two major branches: the innate immune system and adaptive immune system. The innate immune system serves as the first line of defense that protects the host against a variety of threats, ranging from foreign objects (e.g. pathogens) to altered-self (e.g. cancer cells). Similar to a knee jack reaction, it activates extremely quickly, usually within minutes upon microbial encounters. In contrast, the adaptive immune system provides the host with a specific and long-term protection against the aforementioned threats.

Notably, innate immune activation delivers information that facilitates the initiation of the adaptive immune responses. In line with this notion, aberrant activation of the innate immune system has been linked to the etiologies of important human abnormalities such as autoinflammatory diseases and cancer. Therefore, understanding the molecular mechanisms governing innate immune activation is pivotal for harnessing the power of the immune system for therapeutic innovations.

Q) And what is your focus?

At the cellular level, the innate immune system uses distinct pattern recognition receptors (PRR) to sense conserved microbial products or danger signals. The molecular mechanisms of innate immune signal transduction is an area under intensive investigation. However, it is still unclear how these receptors bridge ligand sensing to diverse downstream host protective mechanisms. In order to fill this gap of knowledge, I focus on a family of proteins known as the Toll-like receptors (TLRs) — the prototypical family of PRRs.

TLRs are transmembrane proteins that reside on the plasma and endosomal membranes. They detect a wide range of microbial products, including bacterial lipopolysaccharides (LPS), lipoproteins, flagellin, and nucleic acids. TLR activation induces a myriad of host responses such as metabolic reprogramming, activation of the NFκB pathway, and pro-inflammatory cytokine production. However, whether and how these distinct activities are organized together remains elusive.

The adaptor protein MyD88 is genetically required for TLR-mediated pro-inflammatory cytokine expression, which means that in the absence of this gene, the TLR pathway is largely ablated. Recently structural evidence revealed that MyD88 forms a large protein complex with kinases named IRAK2 and IRAK4 in vitro (also known as the myddosome). It has been proposed that this protein complex may control TLR-induced host responses in living cells, experimental evidence supporting such proposal, however, has remained sparse.

Based on the observations that the myddosome assembles immediately upon TLR stimulation in living cells, I hypothesized that this protein complex may function as an organizing center to orchestrate distinct downstream host responses.

Q) You hypothesized that the myddosome functions as an organizing center. Is that what you found?

Indeed, I discovered that the myddosome is critical for TLR-mediated pro-inflammatory cytokine gene expression and metabolic reprogramming, with the later manifested in the form of rapid glycolysis induction. Surprisingly, I identified that the myddosome induces glycolysis via the kinase TBK1. This discovery is novel in that: 1. TBK1 is a new component of the myddosome; 2. Being the key kinase involved in type I interferon production in other PRR signaling pathways (e.g. TRIF, STING, and RIG-I), TBK1-induced metabolic alteration downstream of the myddosome has broadened the cellular functions control by this kinase. 3. Since TBK1 is not required for TLR-dependent NF-  activation,  my findings suggested that the myddosome serves as a modular signaling center that organizes distinct TLR-mediated activities via different components (TBK1-metabolism; IRAK kinases- NF-  activation/cytokine production).

I further hypothesized that if myddosome is a truly modular signaling organizing center, then one should be able to rewire the functional outcomes of this protein complex beyond those activities selected by nature.

Q) And you tested that idea as well?

Yes, I employed synthetic biology approaches to engineer distinct MyD88 chimera constructs in which MyD88 is fused with a protein motif (from STING) or a protein (RIPK3) critical for type-I interferon production or cell death. Macrophages expressing these synthetic MyD88 molecules produce type I interferon production or undergo cell death upon TLR stimulation. Of note, natural MyD88 signaling does not trigger these responses.  Therefore, I have further demonstrated the modular nature of myddosome signaling by reprogramming the biological outcomes of the complex.

Key to myddosome formation is the ability of MyD88 to form oligomeric structures. Inspired by my previous findings, I speculated that one could possibly design synthetic signaling nanomachines by fusing a small molecule controllable-protein oligomerization platform and an effector protein motif together. The FKBP-STING C-terminal fusion construct was then designed as the prototype to test this hypothesis. In this setting, the small molecule induced protein oligomerization promotes type I interferon production even in the absence of endogenous STING. In summary, I engineered a synthetic nanomachine that can induce interferon responses in response to a chemical ligand, transcending the requirement for a receptor and a microbial product. This is the beginning of the design of user-defined innate immune responses. Furthermore, as protein oligomerization is a common theme observed in other innate immune signaling pathways, such as the inflammasome, the RIG-I, and cGas-STING pathways, my work also opens the possibilities for pathway-specific modulation or engineering.

Q) In other words, this nanomachine can trigger an innate immune response without the presence of a pathogen. What’s the implication of this?

The signaling pathways and proteins of the innate immune system are modular. Based on the wealth of knowledge accumulated from basic researches, we can start to rewire the pathways — just like playing LEGO toys — to engineer controllable, user-defined innate immune responses for revealing new biology as well as developing potential innovative therapeutics.

 Q) What drew you to this work?

I have always been interested in figuring out how things works in general. Trained as a microbiologist during my PhD, I have learned a great deal about the sophisticated tricks that bacteria employ to build home in mammalian host cells. By the end of my graduate training, I was fascinated by ability of the innate immune to immediately respond to different levels of threats. Therefore, for my postdoc training, I decided to come to the other side of the table and to study the spaiotemporal regulation of the innate immune system. I am especially grateful to my mentor Dr. Jonathan Kagan who allows me to explore all sorts of different directions and possibilities in the lab.  In the same vein, the JCC fellowship provides critical support for my postdoctoral training.

Professionally, I found unexpected discoveries made during basic cell biological and biochemical researches most exciting for me.

Q) And when you’re not in the lab?

Personally, I like hiking and exploring the splendid shorelines along MA with my wife. Now as a father of my one-year old daughter, I also like playing with her and reading to her books written by Sandra Boynton such as Moo Baa Lalala.

Thanks, Yunhao Tan!