SUMMARY Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1–88 in the Pin1 interdomain cleft in a disordered, or “fuzzy”, complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1–88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells.

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The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms—important in causing chronic and therapy resistant infections—is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.

Tissue factor pathway inhibitor-2 (TFPI-2) has previously been characterized as an endogenous anticoagulant. TFPI-2 is expressed in the vast majority of cells, mainly secreted into the extracellular matrix. Recently we reported that EDC34, a C-terminal peptide derived from TFPI-2, exerts a broad antimicrobial activity. In the present study, we describe a previously unknown antimicrobial mode of action for the human TFPI-2 C-terminal peptide EDC34, mediated via binding to immunoglobulins of the classes IgG, IgA, IgE, and IgM. In particular the interaction of EDC34 with the Fc part of IgG is of importance since this boosts interaction between the immunoglobulin and complement factor C1q. Moreover, we find that the binding increases the C1q engagement of the antigen-antibody interaction, leading to enhanced activation of the classical complement pathway during bacterial infection. In experimental murine models of infection and endotoxin challenge, we show that TFPI-2 is up-regulated in several organs, including the lung. Correspondingly, TFPI-2−/− mice are more susceptible to pulmonary Pseudomonas aeruginosa bacterial infection. No anti-coagulant role of TFPI-2 was observed in these models in vivo. Furthermore, in vivo, the mouse TFPI-2-derived C-terminal peptide VKG24, a homolog to human EDC34 is protective against systemic Escherichia coli bacterial infection. Moreover, in sputum from cystic fibrosis patients TFPI-2 C-terminal fragments are generated and found associated with immunoglobulins. Together our data describe a previously unknown host defense mechanism and therapeutic importance of TFPI-2 against invading Gram-negative bacterial pathogens.