Revealing the signaling of complement receptors C3aR and C5aR1 by anaphylatoxins.

The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

Molecular insights into DNA recognition and methylation by non-canonical type I restriction-modification systems.

Type I restriction-modification systems help establish the prokaryotic DNA methylation landscape and provide protection against invasive DNA. In addition to classical m6A modifications, non-canonical type I enzymes catalyze both m6A and m4C using alternative DNA-modification subunits M1 and M2. Here, we report the crystal structures of the non-canonical PacII_M1M2S methyltransferase bound to target DNA and reaction product S-adenosylhomocysteine in a closed clamp-like conformation. Target DNA binds tightly within the central tunnel of the M1M2S complex and forms extensive contacts with all three protein subunits. Unexpectedly, while the target cytosine properly inserts into M2's pocket, the target adenine (either unmethylated or methylated) is anchored outside M1's pocket. A unique asymmetric catalysis is established where PacII_M1M2S has precisely coordinated the relative conformations of different subunits and evolved specific amino acids within M2/M1. This work provides insights into mechanisms of m6A/m4C catalysis and guidance for designing tools based on type I restriction-modification enzymes.

Intrinsic Aerobic Capacity Affects Hippocampal pAkt and HSP72 Response to an Acute High Fat Diet and Heat Treatment in Rats.

BACKGROUND: Aerobic capacity is associated with metabolic, cardiovascular, and neurological health. Low-capacity runner (LCR) rats display low aerobic capacity, metabolic dysfuction, and spatial memory deficits. A heat treatment (HT) can improve metabolic dysfunction in LCR peripheral organs after high fat diet (HFD). Little is known about metabolic changes in the brains of these rats following HT. OBJECTIVE: Our objective was to examine the extent to which high or low aerobic capacity impacts Akt (a protein marker of metabolism) and heat shock protein 72 (HSP72, a marker of heat shock response) after HFD and HT in hippocampus. METHODS: We measured phosphorylated Akt (pAkt) in the striatum and hippocampus, and HSP72 in the hippocampus, of HFD-fed and chow-fed LCR and high-capacity runner (HCR) rats with and without HT. RESULTS: pAkt was lower in the hippocampus of chow-fed LCR than HCR rats. HFD resulted in greater pAkt in LCR but not HCR rats, but HT resulted in lower pAkt in the LCR HFD group. HSP72 was greater in both HCR and LCR rat hippocampus after HT. The HFD blunted this effect in LCR compared to HCR hippocampus. CONCLUSION: The abnormal phosphorylation of Akt and diminished HSP response in the hippocampus of young adult LCR rats might indicate early vulnerability to metabolic challenges in this key brain region associated with learning and memory.

Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion.

DNA-induced liquid-liquid phase separation of cyclic GMP-AMP synthase (cGAS) triggers a potent response to detect pathogen infection and promote innate immune signaling. Whether and how pathogens manipulate cGAS-DNA condensation to mediate immune evasion is unknown. We report the identification of a structurally related viral tegument protein family, represented by ORF52 and VP22 from gamma- and alpha-herpesvirinae, respectively, that employs a conserved mechanism to restrict cGAS-DNA phase separation. ORF52/VP22 proteins accumulate into, and effectively disrupt, the pre-formed cGAS-DNA condensation both in vitro and in cells. The inhibition process is dependent on DNA-induced liquid-liquid phase separation of the viral protein rather than a direct interaction with cGAS. Moreover, highly abundant ORF52 proteins carried within viral particles are able to target cGAS-DNA phase separation in early infection stage. Our results define ORF52/VP22-type tegument proteins as a family of inhibitors targeting cGAS-DNA phase separation and demonstrate a mechanism for how viruses overcome innate immunity.

Structural insights into assembly, operation and inhibition of a type I restriction-modification system.

Type I restriction-modification (R-M) systems are widespread in prokaryotic genomes and provide robust protection against foreign DNA. They are multisubunit enzymes with methyltransferase, endonuclease and translocase activities. Despite extensive studies over the past five decades, little is known about the molecular mechanisms of these sophisticated machines. Here, we report the cryo-electron microscopy structures of the representative EcoR124I R-M system in different assemblies (R2M2S1, R1M2S1 and M2S1) bound to target DNA and the phage and mobile genetic element-encoded anti-restriction proteins Ocr and ArdA. EcoR124I can precisely regulate different enzymatic activities by adopting distinct conformations. The marked conformational transitions of EcoR124I are dependent on the intrinsic flexibility at both the individual-subunit and assembled-complex levels. Moreover, Ocr and ArdA use a DNA-mimicry strategy to inhibit multiple activities, but do not block the conformational transitions of the complexes. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into assembly, operation and inhibition mechanisms of type I R-M systems.

Molecular Mechanism of RNA Recognition by Zinc-Finger Antiviral Protein.

Zinc-finger antiviral protein (ZAP) is a host antiviral factor that specifically restricts a wide range of viruses. ZAP selectively binds to CG-dinucleotide-enriched RNA sequences and recruits multiple RNA degradation machines to degrade target viral RNA. However, the molecular mechanism and structural basis for ZAP recognition of specific RNA are not clear. Here, we report the crystal structure of the ZAP N-terminal domain bound to a CG-rich single-stranded RNA, providing the molecular basis for its specific recognition of a CG dinucleotide and additional guanine and cytosine. The four zinc fingers of ZAP adopt a unique architecture and form extensive interactions with RNA. Mutations of both protein and RNA at the RNA-ZAP interacting surface reduce the in vitro binding affinity and cellular antiviral activity. This work reveals the molecular mechanism of ZAP recognition of specific target RNA and also provides insights into the mechanism by which ZAP coordinates downstream RNA degradation processes.

HpoR, a novel c-di-GMP effective transcription factor, links the second messenger's regulatory function to the mycobacterial antioxidant defense.

Cyclic di-GMP (c-di-GMP) is a global signaling molecule that widely modulates diverse cellular processes. However, whether or not the c-di-GMP signal participates in regulation of bacterial antioxidant defense is unclear, and the involved regulators remain to be explored. In this study, we characterized HpoR as a novel c-di-GMP effective transcription factor and found a link between the c-di-GMP signal and the antioxidant regulation in Mycobacterium smegmatis. H2O2 stress induces c-di-GMP accumulation in M. smegmatis. High level of c-di-GMP triggers expression of a redox gene cluster, designated as hpoR operon, which is required for the mycobacterial H2O2 resistance. HpoR acts as an inhibitor of the hpoR operon and recognizes a 12-bp motif sequence within the upstream regulatory region of the operon. c-di-GMP specifically binds with HpoR at a ratio of 1:1. Low concentrations of c-di-GMP stimulate the DNA-binding activity of HpoR, whereas high concentrations of the signal molecule inhibit the activity. Strikingly, high level of c-di-GMP de-represses the intracellular association of HpoR with the regulatory region of the hpoR operon in M. smegmatis and enhances the mycobacterial H2O2 resistance. Therefore, we report a novel c-di-GMP effective regulator in mycobacteria, which extends the second messenger's function to bacterial antioxidant defense.