Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.

IntroductionCD8 ϩ T cells are critical mediators of adaptive inflammatory responses to intracellular pathogens. They require a series of signals for efficient expansion and acquisition of effector functions, such as cytokine secretion and lytic activity. These signals are delivered by professional antigen-presenting cells (APC) and include antigen recognition (signal 1), costimulatory activation (signal 2), and signaling provided by innate inflammatory cytokines (signal 3). 1 Whereas signals 1 and 2 prime naive CD8 ϩ T cells and initiate cell division, signal 3 cytokines program effector functions and ensure clonal survival. A variety of cytokines have the potential to act as signal 3 in CD4 ϩ and CD8 ϩ T cells, 2-4 and interleukin (IL)-12 and interferon (IFN)-␣/␤, in particular, promote efficient induction of innate immunity as well as the development of adaptive type 1 responses to intracellular infection. 5,6 Thus, IL-12 and IFN-␣/␤ may represent the predominant signal 3 during intracellular infection.Whereas IL-12 regulates T helper (Th) 1 development in CD4 ϩ T cells, early reports suggested that the induction of IFN-␥ secretion and lytic activity in CD8 ϩ T cells was independent of IL-12, signal transducer and activator of transcription (STAT) 4, and T-box expressed in T cells (T-bet). [7][8][9] However, recent studies by Schmidt and Mescher found that in vitro priming with IL-12 induced high and sustained secretion of IFN-␥ and markedly enhanced lytic activity in murine CD8 ϩ T cells. 10 Furthermore, these effects were dependent on STAT4, indicating that IL-12 signaling provides a necessary third signal for the regulation of CD8 ϩ T-cell development. 3,11,12 More recent studies have indicated that IFN-␣/␤ can act in a manner similar to IL-12 to provide signal 3 and promote the induction of cytokine secretion, cytolytic activity, and clonal expansion in murine CD8 ϩ T cells. 3,13 Collectively, these studies suggested that IL-12 and IFN-␣ can act as redundant signals to promote the development of effector responses in murine CD8 ϩ T cells.In addition to enhancing effector cell development, IFN-␣/␤ was implicated in the generation of memory CD8 ϩ T cells in vivo. In these studies, IFN-␣/␤ receptor (IFNAR)-deficient, T-cell receptor (TCR)-transgenic (P14) CD8 ϩ T cells failed to expand and generate memory populations in response to in vivo lymphocytic choriomeningitis virus infection despite their ability to proliferate efficiently in vitro. 13 Alternatively, IL-12 Ϫ/Ϫ mice displayed defective primary effector responses, whereas development of T central memory (T CM ) cells was markedly enhanced compared with wild type, indicating that IL-12 signaling suppresses T CM development. [14][15][16] Considering that IFN-␣/␤ has been implicated in effector and memory cell development, it is unclear how this signal regulates both events and whether any of these activities operate in human CD8 ϩ T cells. Furthermore, it is not clear how IL-12 and IFN-␣/␤ signals are integrated to balance effector and memory cell develo...

During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-αβ), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-αβ in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-α and IL-12 can promote STAT4 activation, IFN-α failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-α did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-α-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-α lacks this activity due to its inability to promote sustained T-bet expression.

Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) --> MAPK kinase (MKK) --> MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 -->--> p38 kinase -->--> TonE.