Activation of the Endoplasmic Reticulum Stress Response Impacts the NOD1 Signaling Pathway.

Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition receptor (PRR) responsible for sensing bacterial peptidoglycan fragments. Stimulation of NOD1 leads to a robust innate immune response via activation of the major transcription factor NF-κB. In addition to peptidoglycan sensing, NOD1 and the closely related PRR NOD2 have been linked to inflammation by responding to the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). Here we show that differential ER stress induction renders cells more susceptible to Salmonella enterica serovar Typhimurium infection in a NOD1-dependent manner, measured by increased NF-κB activation and cytokine expression. In HeLa57A cells stably transfected with an NF-κB::luciferase reporter, we show that cells undergoing ER stress induced by thapsigargin display a significant increase in NF-κB activation in response to NOD1 stimulation by C12-iE-DAP (acylated derivative of the iE-DAP dipeptide [gamma-d-glutamyl-meso-diaminopimelic acid]) and the S Typhimurium effector protein SopE. Tunicamycin-induced ER stress had no effect on NOD1-stimulated NF-κB activation. We further show that the mouse intestinal epithelial cell line MODE-K and RAW264.7 macrophages are more responsive to Salmonella infection when treated with thapsigargin but not with tunicamycin. These profound differences between thapsigargin- and tunicamycin-treated cells upon inflammation suggest that different components downstream of the UPR contribute to NOD1 activation. We found that the NOD1-induced inflammatory response is dependent on protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) activation in conjunction with stimulation of the inositol triphosphate receptor (IP3R). Together, these results suggest that differential UPR activation makes cells more responsive to bacterial infections in a NOD1-dependent manner.

The C. elegans rab family: identification, classification and toolkit construction.

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).