NLRC5 Functions beyond MHC I Regulation—What Do We Know So Far?

NLRC5 is a member of the NLR family that acts as a transcriptional activator of MHC class I genes. In line with the function of several related NLR proteins in innate immune responses, there is, however, also ample evidence that NLRC5 contributes to innate and adaptive immune responses beyond the regulation of MHC class I genes. In human and murine cells, for example, NLRC5 was proposed to contribute to inflammatory and type I interferon responses. The role of NLRC5 in these and other cellular processes is hitherto still not well understood and blurred by discrepancies in the reported data. Here, we provide a detailed and critical discussion of the available experimental data on the emerging biological functions of NLRC5 in innate immune responses in men and mice. Better awareness of the multiple roles of NLRC5 will help to define its overall contribution to immune responses and cancer

Control of Morphological Differentiation of Streptomyces coelicolor A3(2) by Phosphorylation of MreC and PBP2

During morphological differentiation of Streptomyces coelicolor A3(2), the sporogenic aerial hyphae are transformed into a chain of more than fifty spores in a highly coordinated manner. Synthesis of the thickened spore envelope is directed by the Streptomyces spore wall synthesizing complex SSSC which resembles the elongasome of rod-shaped bacteria. The SSSC includes the eukaryotic type serine/threonine protein kinase (eSTPK) PkaI, encoded within a cluster of five independently transcribed eSTPK genes (SCO4775-4779). To understand the role of PkaI in spore wall synthesis, we screened a S. coelicolor genomic library for PkaI interaction partners by bacterial two-hybrid analyses and identified several proteins with a documented role in sporulation. We inactivated pkaI and deleted the complete SCO4775-4779 cluster. Deletion of pkaI alone delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 had a more severe defect and produced 18% aberrant spores affected in the integrity of the spore envelope. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinases caused a similar defect. Following co-expression of pkaI with either mreC or pbp2 in E. coli, phosphorylation of MreC and PBP2 was demonstrated and multiple phosphosites were identified by LC-MS/MS. Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis

Effect of overbalancing phosphorylation activity on the integrity of spore envelopes.

<p>Live-dead staining of spore chains of the eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A) revealed the presence of dead spores (red) or spores without DNA (black). In contrast, spore chains of the parental M145 strain (A) only contained viable spores (green). Expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> (B-F) caused a similar sporulation defect in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes was able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Effect of Ser/Thr kinases on the viability of spores (A) and the resistance of germinating spores to vancomycin (B).

<p>Spore chains were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) and observed by fluorescence microscopy. Percentage of viable (green), dead (red) and spores without DNA (black) is given for each strain. Spores of the different strains were plated onto LB agar and filter discs containing 5 μg vancomycin were applied. Whereas, M145 and the <i>pkaI</i> mutant NLΔPkaI were resistant, NLΔ4775–4779 spores showed vancomycin sensitivity, suggesting an impaired spore wall. Vancomycin sensitivity of M145 or NLΔPkaI was also caused by expressing a second copy of each kinase gene, with the exception of <i>pkaI</i>. Supplementation of the agar plates with 3 mM MgCl_2, known to rescue mutants impaired in cell wall synthesis restored vancomycin resistance to all strains. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ.</p

Effect of overbalancing phosphorylation activity on proper sporulation of <i>S</i>. <i>coelicolor</i>.

<p>Phase contrast microscopy of spore chains revealed the presence of aberrant spores (arrows) in eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A). In contrast, spore chains of the parental M145 strain (A) and the complemented mutant NLΔPkaI::pSET-pkaI (E) contain mainly regular ovoid spores. Not only deletion, but also expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> causes a similar sporulation defect (B-F, white arrows) in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes is able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Phosphorylation of PBP2 by PkaI.

<p><i>S</i>. <i>coelicolor pbp2</i> with a S-tag encoding sequence was co-expressed with <i>pkaI</i> in <i>E</i>. <i>coli</i>. PBP2_S-tag was purified under denaturing conditions by affinity chromatography. PkaI with an N-terminal His-tag was purified by Ni-NTA chromatography under native conditions. Purified His_PkaI and PBP2_S-tag proteins (bold letters and underlining indicate, which protein was purified) were separated on an SDS polyacrylamide gel and stained with Coomassie blue (<b>A</b>). Phosphorylated proteins were identified by ProQ Diamond staining. The white arrow indicates auto-phosphorylated His_PkaI, while the black arrow marks phosphorylated PBP2_S-tag. Immunoblotting with Anti-S-tag antibodies confirmed the identity of PBP2_S-tag. Domain architecture of PBP2 and positions of the most likely phosphosites (<b>B</b>). Predicted Pfam domains (PBP dimerization, dark grey, PBP transpeptidase, light grey), a transmembrane helix (TM) and the positions of phosphorylated S/T residues, identified by LC-MS/MS, are indicated.</p