No abstract

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP 1 . X-ray structural studies and theoretical calculations show that apoMP 1 induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and "turn off" diverse membranepermeating molecules besides H4n. In vitro experiments show an apoMP 1 dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP 1 inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage.

Proteins that self-assemble into enclosed polyhedral cages, both naturally and by design, are garnering attention for their prospective utility in the fields of medicine and biotechnology. Notably, their potential for encapsulation and surface display are attractive for experiments that require protection and targeted delivery of cargo. The ability to control their opening or disassembly would greatly advance the development of protein nanocages into widespread molecular tools. Toward the development of protein cages that disassemble in a systematic manner and in response to biologically relevant stimuli, here we demonstrate a modular protein cage system that is opened by highly sequence-specific proteases, based on sequence insertions at strategically chosen loop positions in the protein cage subunits. We probed the generality of the approach in the context of protein cages built using the two prevailing methods of construction: genetic fusion between oligomeric components and (non-covalent) computational interface design between oligomeric components. Our results suggest that the former type of cage may be more amenable than the latter for endowing proteolytically controlled disassembly. We show that a successfully designed cage system, based on oligomeric fusion, is modular with regard to its triggering protease. One version of the cage is targeted by an asparagine protease implicated in cancer and Alzheimer's disease, whereas the second version is responsive to the blood-clotting protease, thrombin. The approach demonstrated here should guide future efforts to develop therapeutic vectors to treat disease states where protease induction or misregulation occurs.

The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization-based flexible docking. We computationally scanned 19 amino acid residues at positions −1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19 × 19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and nonglycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5%, and a false positive rate of 7.3%. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the −1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365, and W331, which modulate the pocket size and specific enzyme–peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide binding suboptimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.