A Ripple Effect During Infection

Researchers have obtained a high-resolution structure of the RIP2 protein complex and detailed its interactions with other immune proteins.

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Like fingerprints at a crime scene, infectious organisms such as bacteria leave traces of their presence in the body. Immune cells pick up on these traces and trigger a sequence of molecular events that eventually allows them to identify and destroy the invading microbe. One important protein involved in this process is the receptor interacting protein-2 (RIP2), which relays the ‘danger’ signal to other proteins inside the immune cell.

To understand how RIP2 orchestrates such intracellular events, scientists from Nanyang Technological University, Singapore (NTU) and A*STAR sought to identify the protein partners of RIP2. They found that for RIP2 to be activated, it must first bind to another protein called the nucleotide-binding oligomerization domain protein (NOD), which comes in two forms (NOD1/2) and is responsible for upstream detection of bacteria.

Thereafter, RIP2 physically attaches itself to other RIP2 molecules—a process termed oligomerization—to form a filamentous helical structure termed the RIPosome. The researchers revealed that a part of each RIP2 protein—what is known as the caspase-
activation-and-recruitment-domain (CARD)—is essential for oligomerization to take place.

The team also used a technique called cryo-electron microscopy to visualize the structure of the RIPosome at a resolution of 4.1 Å resolution. By mutating various parts of NOD1/2 and RIP2 proteins, the researchers were able to show that NOD1/2 specifically approaches and binds to RIP2 in a top-down orientation.

“The correct orientation of the interaction between NOD1/2 and RIP2 will give us a strong indication of the stability and natural biological state of these two proteins during an innate immune response,” explained NTU’s Bin Wu, the corresponding author on the study. Such information is crucial to understanding not only the normal immune response to bacterial infections, but also autoimmune disorders in which the immune system wreaks havoc by attacking the body’s own cells.

“Mutations in NOD1/2 and RIP2 are associated with autoimmune diseases such as Crohn’s disease and lupus. From a pharmacological perspective, peptide inhibitors that bind to the interacting sites of these two proteins can be synthesized, acting as competitive inhibitors for the treatment of autoimmune diseases,” Wu explained.

Going forward, the researchers intend to map out the three-dimensional structure of the NOD1/2–RIP2 complex. They are also interested to find out how NOD1/2-RIP2 signaling may interact with other immune pathways when mounting a defense against invading pathogens.

The A*STAR-affiliated researchers contributing to this research are from the Institute of Medical Biology (IMB) and the Institute of Molecular and Cell Biology (IMCB).