Authors:

Nyla Gillus

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About Paper:

Granzyme A (GrA) is a serine protease that promotes intestinal inflammation and induces apoptosis-like cell death in bacteria-infected cells. Increased levels of GrA have been found in patients with severe infections caused by gram-negative bacteria. Lipopolysaccharide (LPS), an endotoxin located on the outer membrane of Gram-negative bacteria, triggers the production of pro-inflammatory cytokines such as interleukins (ILs) in the presence of GrA. Previous studies have shown that this GrA- and LPS-induced cytokine response is dependent on CD14, suggesting that GrA may be involved in the LPS/Toll-Like Receptor 4 (TLR4) signaling pathway. In this pathway, LPS is first transferred to CD14, which facilitates its recognition by TLR4 and subsequently activates the immune response. However, the mechanism of how GrA interacts with LPS and participates in this signaling transduction remains unclear. The goal of this project is to investigate the binding interaction between GrA and LPS. GrA was cloned into the pET28a plasmid and expressed in E. coli BL21(DE3) cells. The protein was refolded from inclusion bodies and purified via immobilized metal affinity chromatography (IMAC) using a Ni- NTA column. Circular dichroism (CD) spectroscopy was used to assess the secondary structure of GrA, and purified protein was used for crystallization screening. To explore the GrA/LPS interaction, native crystals of GrA will be soaked with bacterial LPS and we will also perform co-crystallization. This study aims to provide structural and mechanistic insights into the role of GrA in modulating the LPS/TLR4 signaling pathway. Keywords: Granzyme A; Lipopolysaccharide

Source:

Purdue University / 2025

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Co-authors:

Nyla Gillus