Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+ human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+ T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins

The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLαs, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (Gsα) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.

Characterization and cell cycle regulation of the related human telomeric proteins Pin2 and TRF1 suggest a role in mitosis

Telomeres are essential for preserving chromosome integrity during the cell cycle and have been specifically implicated in mitotic progression, but little is known about the signaling molecule(s) involved. The human telomeric repeat binding factor protein (TRF1) is shown to be important in regulating telomere length. However, nothing is known about its function and regulation during the cell cycle. The sequence of PIN2, one of three human genes (PIN1-3) we previously cloned whose products interact with the Aspergillus NIMA cell cycle regulatory protein kinase, reveals that it encodes a protein that is identical in sequence to TRF1 apart from an internal deletion of 20 amino acids; Pin2 and TRF1 may be derived from the same gene, PIN2/TRF1. However, in the cell Pin2 was found to be the major expressed product and to form homo- and heterodimers with TRF1; both dimers were localized at telomeres. Pin2 directly bound the human telomeric repeat DNA in vitro, and was localized to all telomeres uniformly in telomerase-positive cells. In contrast, in several cell lines that contain barely detectable telomerase activity, Pin2 was highly concentrated at only a few telomeres. Interestingly, the protein level of Pin2 was highly regulated during the cell cycle, being strikingly increased in G2+M and decreased in G1 cells. Moreover, overexpression of Pin2 resulted in an accumulation of HeLa cells in G2+M. These results indicate that Pin2 is the major human telomeric protein and is highly regulated during the cell cycle, with a possible role in mitosis. The results also suggest that Pin2/TRF1 may connect mitotic control to the telomere regulatory machinery whose deregulation has been implicated in cancer and aging.

Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4+ T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Identification of two novel transmembrane γ-carboxyglutamic acid proteins expressed broadly in fetal and adult tissues

The proline-rich γ-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20–24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.

Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes

The goal of this study was to determine whether β1-adrenergic receptor (AR) and β2-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac β2-AR can activate both Gs and Gi proteins, whereas cardiac β1-AR couples only to Gs. To avoid complicated crosstalk between β-AR subtypes, we expressed β1-AR or β2-AR individually in adult β1/β2-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of β1-AR, but not β2-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, β2-AR (but not β1-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting Gi, Gβγ, or phosphoinositide 3 kinase (PI3K) with pertussis toxin, βARK-ct (a peptide inhibitor of Gβγ), or LY294002, respectively. This indicates that β2-AR activates Akt via a Gi-Gβγ-PI3K pathway. More importantly, inhibition of the Gi-Gβγ-PI3K-Akt pathway converts β2-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, β2-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the Gi-Gβγ-PI3K-Akt signaling pathway.

The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins

The binding of killer cell Ig-like Receptors (KIR) to their Class I MHC ligands was shown previously to be characterized by extremely rapid association and dissociation rate constants. During experiments to investigate the biochemistry of receptor–ligand binding in more detail, the kinetic parameters of the interaction were observed to alter dramatically in the presence of Zn2+ but not other divalent cations. The basis of this phenomenon is Zn2+-induced multimerization of the KIR molecules as demonstrated by BIAcore, analytical ultracentrifugation, and chemical cross-linking experiments. Zn2+-dependent multimerization of KIR may be critical for formation of the clusters of KIR and HLA-C molecules, the “natural killer (NK) cell immune synapse,” observed at the site of contact between the NK cell and target cell.

Segregation of Heterotrimeric G Proteins in Cell Surface Microdomains: Gq Binds Caveolin to Concentrate in Caveolae, whereas Gi and Gs Target Lipid Rafts by Default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (Gi, Gq, Gs, and Gβγ) target discrete cell surface microdomains. Gq specifically concentrates in caveolae, whereas Gi and Gs concentrate much more in lipid rafts marked by GPI-anchored proteins (5′ nucleotidase and folate receptor). Gq, apparently without Gβγ subunits, stably associates with plasmalemmal and cytosolic caveolin. Gi and Gs interact with Gβγ subunits but not caveolin. Gi and Gs, unlike Gq, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize Gq preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, Gq, Gi, and Gs all concentrate in lipid rafts as a complex with Gβγ. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the Gq-caveolin complex without Gβγ for trafficking, signaling, and mechanotransduction are discussed.

Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force

Carbohydrate–protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate–selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate–selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl LewisX (sLeX) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to ≈200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca2+ bond between a single sLeX and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.

Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: A novel mechanism for modulating cell signaling

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor Giα inhibitory to Gβγ stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of Gβγ-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655–662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949–953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, β-arrestin, and Gβ, in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or Gβ antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with Gβ antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, β-arrestin, and Gβ has varying consequences on their ability to associate. As a result, increased availability of and signaling via Gβγ could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing Gβγ signaling and opioid tolerance formation.

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein–protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.