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ABSTRACTThe plasminogen receptor, Plg-RKT, is a unique cell surface receptor that is broadly expressed in cells and tissues throughout the body. Plg-RKT localizes plasminogen on cell surfaces and promotes its activation to the broad-spectrum serine protease, plasmin. In this study, we show that overexpression of Plg-RKT protects mice from high fat diet (HFD)-induced adipose and metabolic dysfunction. During the first 10 weeks on the HFD, the body weights of mice that overexpressed Plg-RKT (Plg-RKT-OEX) were lower than those of control mice (CagRosaPlgRKT). After 10 weeks on the HFD, CagRosaPlgRKT and Plg-RKT-OEX mice had similar body weights. However, Plg-RKT-OEX mice showed a more metabolically favourable body composition phenotype. Plg-RKT-OEX mice also showed improved glucose tolerance and increased insulin sensitivity. We found that the improved metabolic functions of Plg-RKT-OEX mice were mechanistically associated with increased energy expenditure and activity, decreased proinflammatory adipose macrophages and decreased inflammation, elevated brown fat thermogenesis, and higher expression of adipose PPARγ and adiponectin. These findings suggest that Plg-RKT signalling promotes healthy adipose function via multiple mechanisms to defend against obesity-associated adverse metabolic phenotypes.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1—3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine- binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF, type I and type II interferons, interleukin (IL)-1β, IL-18, and β-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.

Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant.

Proteins of the plasminogen activation system are broadly expressed throughout the nervous system, and key roles for these proteins in neuronal function have been demonstrated. Recent reports have established that plasminogen is synthesized in neuroendocrine tissues, making this protein and the proteolytic activity of the product of its activation, plasmin, available at sites separated anatomically from circulating, hepatocyte-derived plasminogen. Results with plasminogen-deficient humans and mice suggest a role for plasminogen in neuritogenesis. To elucidate the role of the plasminogen activation system in these processes, the function of plasminogen during neuritogenesis and neurite outgrowth was studied. It is shown here that plasminogen participates in neuritogenesis, as plasmin inhibitors reduced both neurite outgrowth and neurite length in PC-12 cells. The addition of exogenous plasminogen enhanced neurite outgrowth and neurite length in both PC-12 cells and primary cortical neurons. The proteolytic activity of plasmin was required, since mutation of the catalytic serine residue completely abolished the stimulatory activity. Furthermore, mutation of the lysine binding site within kringle 5 of the plasminogen molecule also reduced the neuritogenic activity of plasminogen. Additionally, we demonstrate that plasminogen specifically bound to laminin-1, the interaction resulted in increased plasminogen activation by tissue-type plasminogen activator, and was dependent on a functional lysine binding site within plasminogen kringle 5. Moreover, during NGF-induced neuritogenesis, laminin-1 was degraded, and this cleavage was catalyzed by plasmin. This study provides the first direct evidence that plasminogen participates in neurite outgrowth and also suggests that laminin-1 degradation by plasmin contributes to the process of neuritogenesis. [ABSTRACT FROM AUTHOR]

The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-γBPs) with homology to the ligand binding domains of the host's IFN- receptor (IFN-γR1). Homology between these proteins is limited to the extracellular portions of the IFN-γR1 and the first 200 amino acids of the IFN-γBPs. The remaining 60 amino acids at the C termini of the IFN-γBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-γBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-γ binding despite having no predicted homology to the IFN-γR1. Truncation of the ECTV IFN-γBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-γ and human IFN-γ (hIFN-γ), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-γBP resulted in comparable loss of hIFN-γ binding activity by SPR. Full-length IFN-γBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-γ with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-γBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-γ binding. [ABSTRACT FROM AUTHOR]

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpδ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects. [ABSTRACT FROM AUTHOR]