Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA "breathing" on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation.

ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62-PB1 and UBA-are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST-tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.

Inflammatory Th1 and Th17 in the Intestine Are Each Driven by Functionally Specialized Dendritic Cells with Distinct Requirements for MyD88.

Normal dynamics between microbiota and dendritic cells (DCs) support modest numbers of T cells, yet these do not cause inflammation. The DCs that induce inflammatory T cells and the signals that drive this process remain unclear. Here, we demonstrate that small intestine DCs lacking the signaling attenuator A20 induce inflammatory T cells and that the signals perceived and antigen-presenting cell (APC) functions are unique for different DC subsets. Thus, although CD103+CD11b- DCs exclusively instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively instruct IL-17+ T cells. Surprisingly, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells. Thus, in disease pathogenesis, specific DCs instruct specific inflammatory T cells.

Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells.

Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and ß-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation.

The role of spartin and its novel ubiquitin binding region in DALIS occurrence.

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains--a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively--have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63-linked ubiquitin chains but does not bind Lys-48-linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR.

Luteinizing hormone receptor-stimulated progesterone production by preovulatory granulosa cells requires protein kinase A-dependent activation/dephosphorylation of the actin dynamizing protein cofilin.

Activation of the LH receptor (LHR) on preovulatory granulosa cells stimulates the cAMP/protein kinase A (PKA) pathway to regulate expression of genes required for ovulation and luteinization. LHR signaling also initiates rearrangement of the actin cytoskeleton. Because disruption of the actin cytoskeleton has been causally linked to steroidogenesis in various cell models, we sought to identify the cellular mechanisms that may modulate reorganization of the actin cytoskeleton and to determine whether cytoskeletal reorganization is required for steroidogenesis. Herein we report that LHR signaling in preovulatory granulosa cells promotes rapid dephosphorylation of the actin-depolymerizing factor cofilin at Ser3 that is dependent on PKA. The LHR-stimulated dephosphorylation of cofilin(Ser3) switches on cofilin activity to bind actin filaments and enhance their dynamics. Basal phosphorylation of cofilin(Ser3) is mediated by active/GTP-bound Rho and downstream protein kinases; LHR signaling promotes a decrease in active/GTP-bound Rho by a PKA-dependent mechanism. LHR-dependent Rho inactivation and subsequent activation of cofilin does not involve ERK, epidermal growth factor receptor, or phosphatidylinositol 3-kinase pathways downstream of PKA. To understand the biological significance of cofilin activation, preovulatory granulosa cells were transduced with a mutant cofilin adenoviral vector in which Ser3 was mutated to Glu (S-E cofilin). Inactive S-E cofilin abolished LHR-mediated reorganization of the actin cytoskeleton and caused a 70% decrease in LHR-stimulated progesterone that is obligatory for ovulation. Taken together, these results show that LHR signaling via PKA activates a cofilin-regulated rearrangement of the actin cytoskeleton and that active cofilin is required to initiate progesterone secretion by preovulatory granulosa cells.

Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D.

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.