Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers

Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 M guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0-8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by oxaloacetate.     

Limited proteolysis study of the structure of chloroplast coupling factor CF1

The structure of chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bonds E17--G18, R21--E22, E22--V23, and K24--V25. The cleavage of peptide bonds at amino acid residues E22 or/and V25 caused weaker subunit interactions and partial dissociation of the alpha- and gamma-subunits. In the N-terminal fragment of the isolated CF1 beta-subunit, the L14--E15 bond was found to be exposed to proteolytic attack. Also, the alphaS86--S87, alphaE125--S126, alphaR127--L128, and betaV76--A77 bonds were subject to proteolysis. The correlation between the accessibility of these bonds to proteases and the surface location of similar bonds in mitochondrial F1 was deduced from its molecular structure and a computed model of CF1. The K204--C205 bond exposed to protease on reduction of the CF1 gamma-subunit was shown to be masked when the catalytic reaction was initiated by CaATP.     

Cutting edge: Membrane nanotubes connect immune cells

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.     

Cutting edge: TRAIL deficiency accelerates hematological malignancies

TNF apoptosis-inducing ligand is attracting considerable interest as a potential extrinsic tumor suppressor mechanism, although previous reports have conveyed somewhat contrasting views regarding the likely importance of this pathway. In this study, we provide the first evaluation of spontaneous tumor formation over the life span of TRAIL-deficient mice. Interestingly, >25% of these mice do develop lymphoid malignancies after 500 days of life. TRAIL suppressed the initiation and development of both tumors of lymphoid and stromal origin in the context of the loss of at least one p53 allele. Specific examination of the role of TRAIL in Her2/neu oncogene-driven mammary epithelial cancer revealed no critical role for TRAIL despite the inherent TRAIL sensitivity of such mammary carcinomas. Overall, the data indicate an important function of TRAIL in controlling carcinogenesis, but suggest that further examination of this pathway in epithelial malignancies is warranted.     

Cutting edge: TREM-2 attenuates macrophage activation

The triggering receptor expressed on myeloid cells 2 (TREM-2) delivers intracellular signals through the adaptor DAP12 to regulate myeloid cell function both within and outside the immune system. The role of TREM-2 in immunity has been obscured by the failure to detect expression of the TREM-2 protein in vivo. In this study, we show that TREM-2 is expressed on macrophages infiltrating the tissues from the circulation and that alternative activation with IL-4 can induce TREM-2. TREM-2 expression is abrogated by macrophage maturation with LPS of IFN-gamma. Using TREM-2(-/-) mice, we find that TREM-2 functions to inhibit cytokine production by macrophages in response to the TLR ligands LPS, zymosan, and CpG. Furthermore, we find that TREM-2 completely accounts for the increased cytokine production previously reported by DAP12(-/-) macrophages. Taken together, these data show that TREM-2 is expressed on newly differentiated and alternatively activated macrophages and functions to restrain macrophage activation.     

Cutting edge commentary: origins of B-1 cells

The origin of B-1a cells, a minority population of B cells that express CD5, are abundant in coelomic cavities, and often produce autoantibodies, has been the subject of study for many years. Accumulating evidence demonstrates that the hypothesis that only B cells arising in fetal or neonatal tissues have the potential to become B-1a cells cannot be true. Rather, B cell receptor-mediated signaling initiated by ligation of autoantigen has now been shown to be required for induction of the B-1a phenotype. Furthermore, cells with a functional B-1a phenotype can be induced from adult precursors by appropriate Ag. At the same time, microenvironment-specific events may determine the likelihood that a given B cell, either adult or fetal derived, enters this pathway. CD5 expression and possibly localization to the peritoneum appear to provide some protection to autoreactive cells otherwise slated for elimination.     

Cutting edge: molecular portrait of human autoimmune disease

Autoimmune diseases affect 3-5% of the population, are mediated by the immune response to self-Ags, and are characterized by the site of tissue destruction. We compared expression levels of >4,000 genes in PBMC of control individuals before and after immunization to those of individuals with four distinct autoimmune diseases. The gene expression profile of the normal immune response exhibits coordinate changes in expression of genes with related functions over time. In contrast, each individual from all autoimmune diseases displays a similar gene expression profile unrelated to the pattern of the immunized group. To our surprise, genes with a distinct expression pattern in autoimmunity are not necessarily "immune response" genes, but are genes that encode proteins involved in apoptosis, cell cycle progression, cell differentiation, and cell migration.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.     

Cutting edge: anti-inflammatory properties of low levels of IFN-gamma

Activation of naive T and B cells occurs only within the context of organized lymphoid tissue. Thus, the continuous recirculation of mature lymphocytes is crucial for the development of primary immune response to foreign Ags. We have previously shown that low levels of IFN-gamma inhibit homing of B cells to the secondary lymphoid organs. In this study, we demonstrate that similarly low doses of IFN-gamma down-regulate integrin-mediated adhesion and migration of naive T and Th2 cells, and have a profound effect on the in vivo homing of naive T cells to the lymph nodes. Moreover, we show that these low doses of IFN-gamma have anti-inflammatory effects in an in vivo asthma model. Thus, in contrast to the proinflammatory effects of IFN-gamma at relatively high concentrations, low dose IFN-gamma appears to exert global suppressory effects on T cell trafficking and may have clinical application as an anti-inflammatory agent.     

Cutting edge: TCR revision occurs in germinal centers

Mouse CD4(+)Vbeta5(+) T cells recognize a peripherally expressed superantigen encoded by an endogenous retrovirus. Ag encounter tolerizes the mature CD4 T cell compartment, either by deletion of autoreactive cells or by TCR revision. This latter process is driven by TCRbeta rearrangement through RAG activity and results in the rescue of cells expressing novel TCRs that no longer recognize the tolerogen. Consistent with the notion that revising T cells represent a distinct peripheral T cell population, we now show that these lymphocyte blasts express a hybrid effector/memory phenotype and are not undergoing cell division. A population of revising T cells is CD40(+), expresses the germinal center (GC) marker CXCR5, and is Vbeta5(low)Thy-1(low). Histology reveals that, consistent with their surface Ag phenotype, T cells undergoing TCR revision are enriched in splenic GCs. These data demonstrate that TCR revision is a multistep tolerance pathway supported by the unique microenvironment provided by GCs.     

Cutting edge: antigen-independent CD8 T cell proliferation

Recent analyses of CD8 T cell responses to Listeria monocytogenes infection demonstrate that the duration of in vivo T cell proliferation is not determined by the amount or duration of Ag presentation. However, the extent to which T lymphocytes are capable of proliferating in the absence of Ag is unknown. Herein we demonstrate that CD8 T lymphocytes undergo up to eight rounds of proliferation in the absence of Ag following transient, 2.5-h in vitro antigenic stimulation. Ag-independent expansion of CD8 T cells is driven by IL-2 and is further augmented by IL-7 or IL-15. These experiments clearly demonstrate that CD8 T cells undergo prolonged proliferation following transient Ag exposure and support the notion that in vivo CD8 T cell expansion following infection can be uncoupled from Ag presentation.     

Cutting edge: TIGIT has T cell-intrinsic inhibitory functions

Costimulatory molecules regulate the functional outcome of T cell activation, and disturbance of the balance between activating and inhibitory signals results in increased susceptibility to infection or the induction of autoimmunity. Similar to the well-characterized CD28/CTLA-4 costimulatory pathway, a newly emerging pathway consisting of CD226 and T cell Ig and ITIM domain (TIGIT) has been associated with susceptibility to multiple autoimmune diseases. In this study, we examined the role of the putative coinhibitory molecule TIGIT and show that loss of TIGIT in mice results in hyperproliferative T cell responses and increased susceptibility to autoimmunity. TIGIT is thought to indirectly inhibit T cell responses by the induction of tolerogenic dendritic cells. By generating an agonistic anti-TIGIT Ab, we demonstrate that TIGIT can inhibit T cell responses directly independent of APCs. Microarray analysis of T cells stimulated with agonistic anti-TIGIT Ab revealed that TIGIT can act directly on T cells by attenuating TCR-driven activation signals.     

Cutting edge: Immunosuppressant as adjuvant for tolerogenic immunization

Vaccination for autoimmune and alloimmune diseases has long been an attractive idea. Yet, there is no suitable adjuvant to forcefully steer the immune response toward tolerance. In this study we show that dexamethasone, a potent glucocorticoid immunosuppressant, can function as a tolerogenic adjuvant when applied together with peptide immunogen. BALB/c mice with pre-established delayed-type hypersensitivity to hen OVA were immunized with an OVA-derived, MHC II-restricted peptide (OVA(323-339)) in the presence of dexamethasone. The treatment caused long-term desensitization in treated animals to hen OVA via a dexamethasone-dependent tolerogenic mechanism that blocks maturation of dendritic cells and expands OVA(323-339)-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells in vivo. Similar treatment of NOD mice using dexamethasone and an insulin-derived, MHC II-restricted peptide (B:9-23) prevented predisposed spontaneous diabetes. Remarkably, in both models, dexamethasone-augmented immunization induced long-term persistent, Ag-specific regulatory T cells responsive to recall Ags. These results reveal for the first time the potential usefulness of immunosuppressants as tolerogenic adjuvants.     

Cutting edge: proangiogenic properties of alternatively activated dendritic cells

Angiogenesis plays an important role in tissue remodeling and repair during the late phase of inflammation. In the present study, we show that human dendritic cells (DC) that matured in the presence of anti-inflammatory molecules such as calcitriol, PGE2, or IL-10 (alternatively activated DC) selectively secrete the potent angiogenic cytokine vascular endothelial growth factor (VEGF) isoforms VEGF165 and VEGF121. No VEGF production was observed in immature or classically activated DC. Also, the capacity to produce VEGF was restricted to the myeloid DC subset. When implanted in the chick embryo chorioallantoic membrane, alternatively activated DC elicit a marked angiogenic response, which is inhibited by neutralizing anti-VEGF Abs and by the VEGFR-2 inhibitor SU5416. Therefore, alternatively activated DC may contribute to the resolution of the inflammatory reaction by promoting VEGF-induced angiogenesis.