Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1–3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP—but not unmodified MDP—constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

No abstract

The NLRP3 inflammasome plays a central role in antimicrobial defense, as well as in sterile inflammatory conditions. NLRP3 activity is governed by two independent signals. The first signal primes NLRP3, allowing it to respond to its activation signal. In the murine system, the mitotic spindle kinase NEK7 has been identified as a crucial factor in relaying the activation signal to NLRP3. Here we show that the requirement for NEK7 can be bypassed by TAK1-dependent post-translational priming. Under pro-inflammatory conditions that activate TAK1, NEK7 was dispensable for NLRP3 inflammasome formation in human and murine cells. Intriguingly, dissecting the NEK7 requirement in iPSC-derived primary human macrophages revealed that this NEK7-independent mechanism constitutes the predominant NLRP3 priming pathway in these cells. In summary, our results suggest that NEK7 functions as an NLRP3 primingrather than activationfactor that can work in synergy or redundancy with other priming pathways to accelerate inflammasome activation.

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re-quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.

Astrocytes have crucial functions in the central nervous system (CNS) and are major players in many CNS diseases. Research on astrocyte-centered diseases requires efficient and well-characterized gene transfer vectors. Vectors derived from the Adeno-associated virus serotype 9 (AAV9) target astrocytes in the brains of rodents and nonhuman primates. A recombinant (r) synthetic peptide-displaying AAV9 variant, rAAV9P1, that efficiently and selectively transduces cultured human astrocytes, has been described previously. Here, it is shown that rAAV9P1 retains astrocyte-targeting properties upon intravenous injection in mice. Detailed analysis of putative receptors on human astrocytes shows that rAAV9P1 utilizes integrin subunits 𝜶v, 𝜷8, and either 𝜷3 or 𝜷5 as well as the AAV receptor AAVR. This receptor pattern is distinct from that of vectors derived from wildtype AAV2 or AAV9. Furthermore, a CRISPR/Cas9 genome-wide knockout screening revealed the involvement of several astrocyte-associated intracellular signaling pathways in the transduction of human astrocytes by rAAV9P1. This study delineates the unique receptor and intracellular pathway signatures utilized by rAAV9P1 for targeting human astrocytes. These results enhance the understanding of the transduction biology of synthetic rAAV vectors for astrocytes and can promote the development of advanced astrocyte-selective gene delivery vehicles for research and clinical applications.