Cytosolic and mitochondrial malic enzyme isoforms differentially control insulin secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.     

CD8+ alpha beta+ T cells that lack surface CD5 antigen expression are a major lymphotactin (XCL1) source in peripheral blood lymphocytes

To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.     

Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

ORP3 splice variants and their expression in human tissues and hematopoietic cells

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.     

Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms

The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H(+) (or exchanged for OH(-)), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H(+) symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.     

Alefacept, an immunomodulatory recombinant LFA-3/IgG1 fusion protein, induces CD16 signaling and CD2/CD16-dependent apoptosis of CD2(+) cells

Alefacept, an immunomodulatory recombinant fusion protein composed of the first extracellular domain of LFA-3 fused to the human IgG1 hinge, C(H)2, and C(H)3 domains, has recently been shown in phase II and III clinical trials to safely reduce disease expression in patients with chronic plaque psoriasis. Alefacept modulates the function of and selectively induces apoptosis of CD2(+) human memory-effector T cells in vivo. We have sought to gain further understanding of the mechanisms of action that influence the biological activity of alefacept and may contribute to its efficacy and patient responsiveness. Specifically evaluated is the ability of alefacept to activate intracellular signals mediated via CD2 and/or Fc gamma RIII (CD16). Experimentation using isoforms of alefacept engineered to have amino acid substitutions in the IgG1 C(H)2 domain that impact Fc gamma R binding indicate that alefacept mediates cognate interactions between cells expressing human CD2 and CD16 to activate cells, e.g., increase extracellular signal-regulated kinase phosphorylation, up-regulate cell surface expression of the activation marker CD25, and induce release of granzyme B. In the systems used, this signaling is shown to require binding to CD2 and CD16 and be mediated through CD16, but not CD2. Experimentation using human CD2-transgenic mice and isoforms of alefacept confirmed the requirement for Fc gamma R binding for detection of the pharmacological effects of alefacept in vivo. Thus alefacept acts as an effector molecule, mediating cognate interactions to activate Fc gamma R(+) cells (e.g., NK cells) to induce apoptosis of sensitive CD2(+) target cells.     

Apoptotic human lymphocytes have diminished CD4 and CD8 receptor expression

We used quantitative multiparameter flow cytometric assays to simultaneously detect viable, apoptotic, and necrotic human peripheral blood mononuclear cells (PBMC) and immunophenotyped lymphocyte subsets within the PBMC. Apoptosis was induced by a spectrum of treatments, including camptothecin, cisplatin, dexamethasone, hyperthermia, staurosporine, and etoposide in anti-CD3 mAb-stimulated cells and by cyclohexamide in both quiescent and stimulated cells; apoptosis in the latter was augmented by anti-fas mAb. We found that CD4(+) and CD8(+) cells were significantly underrepresented in the apoptotic PBMC and that the percentage of CD4(+) and CD8(+) PBMC each markedly decreased as apoptosis increased. This suggested that surface expression of these receptors was lessened on apoptotic CD4(+) and CD8(+) cells. This was directly confirmed by observation of sorted CD4(+) PBMC. This analysis of a wide variety of apoptotic stimuli demonstrates that diminished CD4 and CD8 surface receptor expression is a common feature of human T lymphocyte apoptosis.     

A single human myosin light chain kinase gene (MLCK; MYLK)

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.     

The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.     

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.     

Environmental and genetic influences on flight metabolic rate in the honey bee,Apis mellifera

The four isoforms of the catalytic subunit of Na(+)/K(+)-ATPase identified in rats differ in their affinities for ions and ouabain. Moreover, its expression is tissue-specific, developmentally and hormonally regulated. The aim of the present work was to evaluate the influence of age on the ratio and density of these isoforms in crude membrane preparations from rat brain hemispheres, brainstem, heart ventricles and kidneys. In all tissues investigated, Na(+)/K(+)-ATPase activity was higher in adults than in neonates but brain tissues presented the most remarkable differences. In these tissues, ouabain inhibition curves for Na(+)/K(+)-ATPase activity revealed the presence of two processes with different sensitivities to ouabain. An increase of approximately sixfold in the expression of the high affinity isoforms was observed between newborn and adult rats. In contrast, the low affinity isoform increased only approximately twofold in brainstem whereas it increased ninefold in brain hemispheres. Unlike brain tissues, a decrease (almost fourfold) in the number of high affinity ouabain binding sites was observed during ontogenesis of the heart. Although limited by the inability to resolve alpha(2) and alpha(3) isoforms, present data indicate that the influence of development on the expression of Na(+)/K(+)-ATPase depends not only on the isoform, but also on the tissue where the enzyme is expressed.     

Activation of T cells through a T cell-specific epitope of CD45.

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.     

Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.     

TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host.     

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.