Structure,Function, and Dynamics of the Gα Binding Domain of Ric-8A

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IKKα Regulates the Mitotic Phase of the Cell Cycle by Modulating Aurora A Phosphorylation

The IKK complex includes two catalytic components, IKKα and IKKβ, in addition to the scaffold protein IKKγ/NEMO. Even though IKKα and IKKβ share significant sequence homology, they have distinct biological roles with IKKβ regulates the classical pathway of NF-κB activation and IKKα regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKα, but not IKKβ, has role in regulating the M phase of the cell cycle. IKKα siRNA knock-down resulted in increased numbers of cells in the G2/M phase of the cell cycle as compared to control and IKKβ siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKα in the regulation of M phase regulatory factors. IKKα was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKα regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

A Kaposi's Sarcoma-Associated Herpesvirus MicroRNA and Its Variants Target the Transforming Growth Factor β Pathway To Promote Cell Survival

Transforming growth factor β (TGF-β) signaling regulates cell growth and survival. Dysregulation of the TGF-β pathway is common in viral infection and cancer. Latent infection by Kaposi''s sarcoma-associated herpesvirus (KSHV) is required for the development of several AIDS-related malignancies, including Kaposi''s sarcoma and primary effusion lymphoma (PEL). KSHV encodes more than two dozen microRNAs (miRs) derived from 12 pre-miRs with largely unknown functions. In this study, we show that miR variants processed from pre-miR-K10 are expressed in KSHV-infected PEL cells and endothelial cells, while cellular miR-142-3p and its variant miR-142-3p_-1_5, which share the same seed sequence with miR-K10a_ +1_5, are expressed only in PEL cells and not in uninfected and KSHV-infected TIME cells. KSHV miR-K10 variants inhibit TGF-β signaling by targeting TGF-β type II receptor (TβRII). Computational and reporter mutagenesis analyses identified three functional target sites in the TβRII 3′ untranslated region (3′UTR). Expression of miR-K10 variants is sufficient to inhibit TGF-β-induced cell apoptosis. A suppressor of the miRs sensitizes latent KSHV-infected PEL cells to TGF-β and induces apoptosis. These results indicate that miR-K10 variants manipulate the TGF-β pathway to confer cells with resistance to the growth-inhibitory effect of TGF-β. Thus, KSHV miRs might target the tumor-suppressive TGF-β pathway to promote viral latency and contribute to malignant cellular transformation.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

Location and Dynamics of the Immunodominant CD8 T Cell Response to SIVΔnef Immunization and SIVmac251 Vaginal Challenge

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.     

Buffon, Darwin, and the Non-Individuality of Species – A Reply to Jean Gayon

Gayon's recent claim that Buffon developed a concept of species as physical individuals is critically examined and rejected. Also critically examined and rejected is Gayon's more central thesis that as a consequence of his analysis of Buffon's species concept, and also of Darwin's species concept, it is clear that modern evolutionary theory does not require species to be physical individuals. While I agree with Gayon's conclusion that modern evolutionary theory does not require species to be physical individuals, I disagree with his reasons and instead provide logical rather than historical reasons for the same conclusion.     

Nonclassical Transpeptidases of Mycobacterium tuberculosis Alter Cell Size,Morphology, the Cytosolic Matrix,Protein Localization,Virulence, and Resistance to β-Lactams

Virtually all bacteria possess a peptidoglycan layer that is essential for their growth and survival. The β-lactams, the most widely used class of antibiotics in human history, inhibit d,d-transpeptidases, which catalyze the final step in peptidoglycan biosynthesis. The existence of a second class of transpeptidases, the l,d-transpeptidases, was recently reported. Mycobacterium tuberculosis, an infectious pathogen that causes tuberculosis (TB), is known to possess as many as five proteins with l,d-transpeptidase activity. Here, for the first time, we demonstrate that loss of l,d-transpeptidases 1 and 2 of M. tuberculosis (Ldt_Mt1 and Ldt_Mt2) alters cell surface morphology, shape, size, organization of the intracellular matrix, sorting of some low-molecular-weight proteins that are targeted to the membrane or secreted, cellular physiology, growth, virulence, and resistance of M. tuberculosis to amoxicillin-clavulanate and vancomycin.     

Comparison of the pulmonary vascular response to prostaglandin A1, prostaglandin F2α and angiotensin II; Metabolism of PGA1 in lung

A rabbit lung preparation, perfused $\text{in vitro}$, was used to examine pulmonary metabolism of prostaglandin A₁ (PGA₁) and to compare the vasoconstrictor actions of PGA₁, prostaglandin F_2α (PGF_2α) and angiotensin II. PGF_2α caused significantly more, and angiotensin II significantly less, vasoconstriction than did an equimolar concentration of PGA₁. Of three likely PGA₁ metabolites only 15-keto-PGA₁ had any significant vasoconstrictor action. Furosemide and aminophylline (10^?3 M) reduced PGA₁, PGF_2α or angiotensin II-induced vasoconstruction. Diphloretin phosphate potentiated the vascular effect of angiotensin II. Furosemide (10^?3 M) and DPP (9.5 × 10^?6 M) significantly reduced pulmonary metabolism of PGA₁ while aminophylline (10^?3 M) had no effect on this process. Perfusion of the lungs with a hypoxic medium had no effect on PGA₁ metabolism.     

Meprinα Transactivates the Epidermal Growth Factor Receptor (EGFR) via Ligand Shedding,thereby Enhancing Colorectal Cancer Cell Proliferation and Migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.     

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rβ/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of β2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of β2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.     

Inhibition of Keratinocyte Differentiation by the Synergistic Effect of IL-17A,IL-22, IL-1α, TNFα and Oncostatin M

Keratinocyte differentiation program leading to an organized epidermis plays a key role in maintaining the first line of defense of the skin. Epidermal integrity is regulated by a tight communication between keratinocytes and leucocytes, particularly under cytokine control. Imbalance of the cytokine network leads to inflammatory diseases such as psoriasis. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokine and antimicrobial-peptide expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocyte differentiation markers, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. In addition, IL-22 and OSM induced epidermal hyperplasia in vitro and M5 induced epidermal thickening and decreased differentiation marker expression in a mouse model, as observed in human psoriatic skin lesions. This study highlights the precise role of cytokines in the skin inflammatory response. IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss while IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity.     

To speciate,or not to speciate? Resource heterogeneity,the subjectivity of similarity,and the macroevolutionary consequences of niche‐width shifts in plant‐feeding insects

Coevolutionary studies on plants and plant‐feeding insects have significantly improved our understanding of the role of niche shifts in the generation of new species. Evolving plant lineages essentially constitute moving islands and archipelagoes in resource space, and host shifts by insects are usually preceded by colonizations of novel resources. Critical to hypotheses concerning ecological speciation is what happens immediately before and after colonization attempts: if an available plant is too similar to the current host(s), it simply will be incorporated into the existing diet, but if it is too different, it will not be colonized in the first place. It thus seems that the probability of speciation is maximized when alternative hosts are at an ‘intermediate’ distance in resource space. In this review, I wish to highlight the possibility that resource similarity and, thus, the definition of ‘intermediate’, are subjective concepts that depend on the herbivore lineage's tolerance to dietary variation. This subjectivity of similarity means that changes in tolerance can either decrease or increase speciation probabilities depending on the distribution of plants in resource space: insect lineages with narrow tolerances are likely to speciate by ‘island‐hopping’ on young, species‐rich plant groups, whereas more generalized lineages could speciate by shifting among resource archipelagoes formed by higher plant taxa. Repeated and convergent origins of traits known to broaden or to restrict host‐plant use in multiple different insect groups provide opportunities for studying how tolerance and resource heterogeneity may interact to determine speciation rates.