Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells.

Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.

Quantitative Proteomics Reveals the Dynamics of Protein Phosphorylation in Human Bronchial Epithelial Cells during Internalization, Phagosomal Escape, and Intracellular Replication of Staphylococcus aureus.

Internalization of Staphylococcus aureus by nonprofessional phagocytic cells is a major suspected cause of persistent and difficult-to-treat infections, including pneumonia. In this study, we established an infection model with 16HBE14o- human bronchial epithelial cells and demonstrated internalization, escape from phagosomal clearance, and intracellular replication of S. aureus HG001 within the first 4 h postinfection. We used quantitative phosphoproteomics to identify characteristic signaling networks in the host at different infection stages. Although we found only minor changes in protein abundance, the infection was accompanied by highly dynamic alterations in phosphorylation events primarily in proteins that are associated with pathways of cytoskeleton dynamics, cell-cell and cell-matrix contacts, vesicle trafficking, autophagy, and GTPase signaling. Analyses of host protein kinases by kinase-substrate mapping, active regulatory site immunoblotting, and prediction algorithms highlighted known and novel host kinases with putative critical roles in S. aureus infection-accompanied signaling including FAK, PKA, PKC, and CDK. Targeted pharmacological inhibition of these kinases resulted in a significant reduction of intracellular S. aureus cells. The current study constitutes a valuable resource for better understanding the infection-relevant molecular pathomechanisms of airway cells and for developing novel host-centric anti-infective strategies for treating S. aureus infections.

Fold and function of the InlB B-repeat.

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the ß-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met.